LOCUS Exported 7621 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AAV expression of Luciferase and EGFP from CMV promoter. ACCESSION . VERSION . KEYWORDS pAAV.CMV.Luc.IRES.EGFP.SV40 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7621) TITLE AAV plasmids REFERENCE 2 (bases 1 to 7621) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7621 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 72..93 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 108..138 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 146..162 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 151..173 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 170..186 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 170..186 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" repeat_region 224..353 /label=AAV2 ITR enhancer 551..930 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 931..1134 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 1085..1105 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 1131..1155 /label=LNCX /note="Human CMV promoter, forward primer" intron 1270..1402 /label=chimeric intron /note="chimera between introns from human beta-globin and immunoglobulin heavy chain genes" primer_bind 1385..1404 /label=pMT2-F /note="Synthetic intron, forward primer" promoter 1447..1465 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1613..3265 /codon_start=1 /gene="luc+" /product="firefly luciferase" /label=luciferase /note="enhanced luc+ version of the luciferase gene" /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV" primer_bind complement(1679..1697) /label=LucNrev /note="Firefly luciferase, reverse primer" misc_feature 3280..3866 /label=IRES2 /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(3447..3464) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 3674..3693 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 3867..4586 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 3867..4583 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(3912..3933) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4173..4192) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4520..4541 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 4611..4732 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(4622..4641) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 4676..4695 /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 4894..5023 /label=AAV2 ITR primer_bind complement(5042..5059) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5042..5058) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5051..5073) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5200..5655 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5287..5306) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5497..5518 /label=F1ori-F /note="F1 origin, forward primer" promoter 5681..5785 /gene="bla" /label=AmpR promoter CDS 5786..6646 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(6004..6023) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6817..7405 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7306..7325 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7559..7576 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 61 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 121 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 181 tatgaccatg attacgccag atttaattaa ggccttaatt aggctgcgcg ctcgctcgct 241 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt 301 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt ccttgtagtt 361 aatgattaac ccgccatgct acttatctac gtagccatgc tctaggaaga tcttcaatat 421 tggccattag ccatattatt cattggttat atagcataaa tcaatattgg ctattggcca 481 ttgcatacgt tgtatctata tcataatatg tacatttata ttggctcatg tccaatatga 541 ccgccatgtt ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta 601 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc 661 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 721 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg 781 gcagtacatc aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa 841 tggcccgcct ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac 901 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg 961 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg 1021 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg 1081 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta 1141 gtgaaccgtc agatcactag aagctttatt gcggtagttt atcacagtta aattgctaac 1201 gcagtcagtg cttctgacac aacagtctcg aacttaagct gcagaagttg gtcgtgaggc 1261 actgggcagg taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc 1321 ttgtcgagac agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc 1381 cactttgcct ttctctccac aggtgtccac tcccagttca attacagctc ttaaggctag 1441 agtacttaat acgactcact ataggctagc ctcgagaatt ctgcagcgcg acttcctggt 1501 cgaatggcgc cgcgtgagta aggccccgga ggccctcttc tttgttcagt tcgagaaggg 1561 ggaaagctac tttcacctgc acgttctggt cgagaccacg ggggtcaagt ccatggaaga 1621 cgccaaaaac ataaagaaag gcccggcgcc attctatccg ctggaagatg gaaccgctgg 1681 agagcaactg cataaggcta tgaagagata cgccctggtt cctggaacaa ttgcttttac 1741 agatgcacat atcgaggtgg acatcactta cgctgagtac ttcgaaatgt ccgttcggtt 1801 ggcagaagct atgaaacgat atgggctgaa tacaaatcac agaatcgtcg tatgcagtga 1861 aaactctctt caattcttta tgccggtgtt gggcgcgtta tttatcggag ttgcagttgc 1921 gcccgcgaac gacatttata atgaacgtga attgctcaac agtatgggca tttcgcagcc 1981 taccgtggtg ttcgtttcca aaaaggggtt gcaaaaaatt ttgaacgtgc aaaaaaagct 2041 cccaatcatc caaaaaatta ttatcatgga ttctaaaacg gattaccagg gatttcagtc 2101 gatgtacacg ttcgtcacat ctcatctacc tcccggtttt aatgaatacg attttgtgcc 2161 agagtccttc gatagggaca agacaattgc actgatcatg aactcctctg gatctactgg 2221 tctgcctaaa ggtgtcgctc tgcctcatag aactgcctgc gtgagattct cgcatgccag 2281 agatcctatt tttggcaatc aaatcattcc ggatactgcg attttaagtg ttgttccatt 2341 ccatcacggt tttggaatgt ttactacact cggatatttg atatgtggat ttcgagtcgt 2401 cttaatgtat agatttgaag aagagctgtt tctgaggagc cttcaggatt acaagattca 2461 aagtgcgctg ctggtgccaa ccctattctc cttcttcgcc aaaagcactc tgattgacaa 2521 atacgattta tctaatttac acgaaattgc ttctggtggc gctcccctct ctaaggaagt 2581 cggggaagcg gttgccaaga ggttccatct gccaggtatc aggcaaggat atgggctcac 2641 tgagactaca tcagctattc tgattacacc cgagggggat gataaaccgg gcgcggtcgg 2701 taaagttgtt ccattttttg aagcgaaggt tgtggatctg gataccggga aaacgctggg 2761 cgttaatcaa agaggcgaac tgtgtgtgag aggtcctatg attatgtccg gttatgtaaa 2821 caatccggaa gcgaccaacg ccttgattga caaggatgga tggctacatt ctggagacat 2881 agcttactgg gacgaagacg aacacttctt catcgttgac cgcctgaagt ctctgattaa 2941 gtacaaaggc tatcaggtgg ctcccgctga attggaatcc atcttgctcc aacaccccaa 3001 catcttcgac gcaggtgtcg caggtcttcc cgacgatgac gccggtgaac ttcccgccgc 3061 cgttgttgtt ttggagcacg gaaagacgat gacggaaaaa gagatcgtgg attacgtcgc 3121 cagtcaagta acaaccgcga aaaagttgcg cggaggagtt gtgtttgtgg acgaagtacc 3181 gaaaggtctt accggaaaac tcgacgcaag aaaaatcaga gagatcctca taaaggccaa 3241 gaagggcgga aagatcgccg tgtaattcta ggggatccgc ccctctccct cccccccccc 3301 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt 3361 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt 3421 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt 3481 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct 3541 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt 3601 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt 3661 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa 3721 ggtaccccat tgtatgggat ctgatctggg gcctcggtac acatgcttta catgtgttta 3781 gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa 3841 aacacgatga taatatggcc acaaccatgg tgagcaaggg cgaggagctg ttcaccgggg 3901 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg 3961 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg 4021 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct 4081 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag 4141 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg 4201 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca 4261 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct 4321 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca 4381 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg 4441 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc 4501 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc 4561 tcggcatgga cgagctgtac aagtaaagcg gccgcttcga gcagacatga taagatacat 4621 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 4681 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa 4741 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt tttaaagcaa 4801 gtaaaacctc tacaaatgtg gtaaaatcga taaggatctt cctagagcat ggctacgtag 4861 ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca 4921 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 4981 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagccttaat taacctaatt 5041 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 5101 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 5161 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat 5221 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 5281 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 5341 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 5401 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 5461 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 5521 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 5581 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 5641 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 5701 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 5761 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 5821 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 5881 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 5941 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 6001 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 6061 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 6121 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 6181 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 6241 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 6301 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 6361 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 6421 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 6481 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 6541 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 6601 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 6661 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 6721 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 6781 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 6841 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 6901 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 6961 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 7021 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 7081 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 7141 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 7201 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 7261 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 7321 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 7381 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 7441 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 7501 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag 7561 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg 7621 t //