pCL-Eco载体

逆病毒包装载体pCL-Eco的使用方法293T Growth Medium:   450 ml DMEM (JRH Biosciences)   50 ml FBS (JRH Sciences)   5 ml Glutamine (200 mM in saline, JRH Sciences)   2.5 ml Gentamycin (10 mg/ml in water, Sigma)HC11 Growth Medium:  5 ml L-Glutamine (200 mM in saline, JRH Sciences)  2.5 ml Bovine Insulin (1 mg/ml in 0.01 N HCL, Sigma)  2.5 ml Gentamycin (10 mg/ml in water, Sigma)  0.5 ml EGF (10 μg/ml in water, Gibco)  50 ml FBS (JRH Sciences)  440 ml RPMI (JRH Sciences)Day 0: Split 293T cells for transfection:  Wash cells off plate with 10 ml Hanks and put into 15 ml Falcon tube  Add 1 ml 10X trypsin and mix by inversion for about 1-2 minutes  Spin at 1000 rpm in clinical centrifuge for 1-2 minutes  Resuspend cells in 10 ml growth medium  Count cells  Plate cells at 1X106 cells per 100 mm dishDay 2: Transfect 293T cells:  Plan transfection using 2 μg total DNA and 12 μl FuGene in 200 μl total   volume per 100 mm dish  Dilute Fugene: 12 μl per reaction in serum free DMEM for total volume of  200 μl  Mix DNA plasmids in polypropelene tubes (Falcon #2063):     1 μg DNA of interest     1 μg pCL-Eco     Add 200 μl diluted FuGene dropwise to each DNA tube  Incubate RT 15 min  Feed cells with 5 ml fresh growth media and add FuGene/DNA mixture to media Day 3: Change medium:  • Remove viral medium and CAREFULLY feed with 6 ml growth medium  This step may reduce a cytostatic factor (produced after transfections) that  can inhibit growth and infection of your target cells.  Split HC11 cells for infection  Seed HC11 cells at 300,000 cells per 100 mm dish  If using coverslips, incubate with FBS for at least 1 hour prior to platingDay 4: Infect target (HC11) cells:  • Collect virus-containing media off 293T cells  • Syringe filter media through .45 μm filter onto target cells   (Evaporation O/N and loss in filter leaves about 5 ml per plate)  • Add 10 μl 5mg/ml polybrene (final conc = 10 μg polybrene/ml media)   (Store polybrene in aliquots to decrease freeze/thaw cycles)  • Wrap plates in parafilm  • Spin cells in clinical centrifuge 10 min at 1800 rpm  • Rotate plate 1/3. Spin 10 min at 1800 rpm  • Rotate plate 1/3. Spin 10 min at 1800 rpm  • Remove viral media and replace with fresh growth mediaDay 6+: Harvest HC11 cells:  • Wash cells with HBSS  • Scrape remaining cells, pellet and flash freeze for protein/RNA analysis  Stain cells for beta-gal expression:  Fixing Solution :    1.35 ml 37% Formaldehyde    0.2 ml Gluteraldehyde    1X PBS to 25 mls  Staining Solution:    625μl 40 mg/ml X-gal in DMF    50 μl 1M MgCL2    750 μl 100 mM Potassium Ferricyanide    750 μl 100 mM Potassium Ferrocyanide    1X PBS to 25 mls    [100 mM Potassium Ferrocyanide = 2.11 g/50ml PBS]    [100 mM Potassium Ferricyanide = 1.64 g/50 ml PBS]  Store at room temperature, protect from light.    1. Make -gal solution and warm in 37°C water bath to prevent crystallization    2. Aspirate off media    3. Wash cells 1X PBS    4. Fix cells 5 min at RT in fixing solution    5. Wash 3X PBS    6 Optional: Add PBS to cells and warm plate at 37°C 5 min (reduces      crystallization)    7. Stain cells with staining solution at 37°C for 1-24 hrs    8. Store stained cells in 70% EtOH at 4°C      (EMBO J. 5: pg 3133, 1986)Note:   HC11 and MEC primary culture cells are very adherent and can withstand   the force of spinning the plates at 1800 rpm. We found that HC11 cells died at   2200 rpm. However, this is cell type-dependent, and spinning speed should be   optimized accordingly.   Expect to see -gal staining at the periphery of the plate. During the spin   infection the media is pushed to the outside. The cells in the center of the plate   will not be exposed to virus and will not be efficiently infected with retrovirus.

LOCUS       pCL-Eco Sequencing Result	12341 bp 	DNA	SYNDEFINITION  pCL-Eco Sequencing ResultACCESSION   KEYWORDS    SOURCE        ORGANISM  other sequences; artificial sequences; vectors.FEATURES             Location/Qualifiers     source          1..12341                     /organism="pCL-Eco Sequencing Result"                     /mol_type="other DNA"     rep_origin      267..886                     /label="pBR322_origin"     misc_feature    complement(1160..1179)                     /label="SV40pro_F_primer"     rep_origin      complement(1164..1241)                     /label="SV40_origin"     terminator      complement(1709..1828)                     /label="SV40_PA_terminator"     misc_feature    complement(1723..1742)                     /label="EBV_rev_primer"     misc_feature    complement(2253..2300)                     /label="SV40_3_splice"     misc_feature    complement(2306..2321)                     /label="SV40_int"     misc_feature    2540..2562                     /label="pGEX_3_primer"     CDS             complement(3033..5030)                     /label="ORF frame 1"                     /translation="MARSTLSKPLKNKVNPRGPLIPLILLMLRGVSTASPGSSPHQVY                     NITWEVTNGDRETVWATSGNHPLWTWWPDLTPDLCMLAHHGPSYWGLEYQSPFSSPPG                     PPCCSGGSSPGCSRDCEEPLTSLTPRCNTAWNRLKLDQTTHKSNEGFYVCPGPHRPRE                     SKSCGGPDSFYCAYWGCETTGRAYWKPSSSWDFITVNNNLTSDQAVQVCKDNKWCNPL                     VIRFTDAGRRVTSWTTGHYWGLRLYVSGQDPGLTFGIRLRYQNLGPRVPIGPNPVLAD                     QQPLSKPKPVKSPSVTKPPSGTPLSPTQLPPAGTENRLLNLVDGAYQALNLTSPDKTQ                     ECWLCLVAGPPYYEGVAVLGTYSNHTSAPANCSVASQHKLTLSEVTGQGLCIGAVPKT                     HQALCNTTQTSSRGSYYLVAPTGTMWACSTGLTPCISTTILNLTTDYCVLVELWPRVT                     YHSPSYVYGLFERSNRHKREPVSLTLALLLGGLTMGGIAAGIGTGTTALMATQQFQQL                     QAAVQDDLREVEKSISNLEKSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYA                     DHTGLVRDSMAKLRERLNQRQKLFESTQGWFEGLFNRSPWFTTLISTIMGPLIVLLMI                     LLFGPCILNRLVQFVKDRISVVQALVLTQQYHQLKPIEYEP*"     CDS             complement(4970..8242)                     /label="ORF frame 2"                     /translation="MGPMGQPLQVLTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAW                     AETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQ                     SPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVL                     DLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL                     ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ                     VKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAP                     LYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL                     TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHA                     VEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD                     ILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAG                     TSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKN                     KDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL                     IENSSPYTSEHFHYTVTDIKDLTKLGAIYDKTKKYWVYQGKPVMPDQFTFELLDFLHQ                     LTHLSFSKMKALLERSHSPYYMLNRDRTLKNITETCKACAQVNASKSAVKQGTRVRGH                     RPGTHWEIDFTEIKPGLYGYKYLLVFIDTFSGWIEAFPTKKETAKVVTKKLLEEIFPR                     FGMPQVLGTDNGPAFVSKVSQTVADLLGIDWKLHCAYRPQSSGQVERMNRTIKETLTK                     LTLATGSRDWVLLLPLALYRARNTPGPHGLTPYEILYGAPPPLVNFPDPDMTRVTNSP                     SLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWK                     GPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLTWRVQRSQNPLKIRLTRE                     AP*"     misc_feature    complement(5079..5095)                     /label="pBMN_5_primer"                     /translation="MGPMGQPLQVLTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAW                     AETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQ                     SPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVL                     DLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL                     ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ                     VKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAP                     LYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVL                     TQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHA                     VEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLD                     ILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAG                     TSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKN                     KDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL                     IENSSPYTSEHFHYTVTDIKDLTKLGAIYDKTKKYWVYQGKPVMPDQFTFELLDFLHQ                     LTHLSFSKMKALLERSHSPYYMLNRDRTLKNITETCKACAQVNASKSAVKQGTRVRGH                     RPGTHWEIDFTEIKPGLYGYKYLLVFIDTFSGWIEAFPTKKETAKVVTKKLLEEIFPR                     FGMPQVLGTDNGPAFVSKVSQTVADLLGIDWKLHCAYRPQSSGQVERMNRTIKETLTK                     LTLATGSRDWVLLLPLALYRARNTPGPHGLTPYEILYGAPPPLVNFPDPDMTRVTNSP                     SLQAHLQALYLVQHEVWRPLAAAYQEQLDRPVVPHPYRVGDTVWVRRHQTKNLEPRWK                     GPYTVLLTTPTALKVDGIAAWIHAAHVKAADPGGGPSSRLTWRVQRSQNPLKIRLTRE                     AP*"     CDS             complement(8570..10186)                     /label="ORF frame 2"                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"     misc_feature    complement(9770..10177)                     /label="gag"                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"     misc_feature    complement(9783..9799)                     /label="pBABE_5_primer"                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"     misc_feature    complement(9815..9837)                     /label="MSCV_primer"                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"     misc_feature    complement(9815..9837)                     /label="pLXSN_5_primer"                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"     misc_feature    complement(10319..11018)                     /label="5_LTR"                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"     promoter        complement(10497..11077)                     /label="CMV_immearly_promoter"                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"     misc_feature    complement(10524..10544)                     /label="CMV_fwd_primer"                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"     misc_feature    complement(10710..10997)                     /label="CAG_enhancer"                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"     misc_feature    11342..11364                     /label="pGEX_3_primer"                     /translation="MGQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWP                     TFNVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVH                     PKPPPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGAKPKPQVLSDSGGPLIDLLTE                     DPPPYRDPRPPPSDRDGNGGEATPAGEAPDPSPMASRLRGRREPPVADSTTSQAFPLR                     AGGNGQLQYWPFSSSDLYNWKNNNPSFSEDPGKLTALIESVLITHQPTWDDCQQLLGT                     LLTGEEKQRVLLEARKAVRGDDGRPTQLPNEVDAAFPLERPDWDYTTQAGRNHLVHYR                     QLLLAGLQNAGRSPTNLAKVKGITQGPNESPSAFLERLKEAYRRYTPYDPEDPGQETN                     VSMSFIWQSAPDIGRKLERLEDLKNKTLGDLVREAEKIFNKRETPEEREERIRRETEE                     KEERRRTEDEQKEKERDRRRHREMSKLLATVVSGQKQDRQGGERRRSQLDRDQCAYCK                     EKGHWAKDCPKKPRGPRGPRPQTSLLTLDD*"     promoter        11523..11551                     /label="AmpR_promoter"