LOCUS Exported 7368 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Cre dependent GFP expression.. ACCESSION . VERSION . KEYWORDS pAAV-CAG-Flex-Rev-3xGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7368) TITLE AAV expression plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 7368) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7368 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(11..28) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(182..770) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(262..281) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(941..1801) /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 1564..1583 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(1802..1906) /gene="bla" /label=AmpR promoter rep_origin complement(1932..2387) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2069..2090) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 2281..2300 /label=F1ori-R /note="F1 origin, reverse primer" repeat_region 2459..2588 /label=AAV2 ITR promoter 2901..3178 /label=chicken beta-actin promoter primer_bind 3458..3480 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 3542..3561 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" protein_bind complement(3596..3629) /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind complement(3692..3725) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS complement(3776..4489) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS complement(4490..4543) /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS complement(4553..5266) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS complement(5267..5323) /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS complement(5333..6049) /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS complement(5333..6049) /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(5375..5396) /label=EGFP-C /note="EGFP, forward primer" primer_bind 5724..5743 /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 5983..6004 /label=EGFP-N /note="EGFP, reverse primer" protein_bind 6091..6124 /label=lox2272 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GGATACTT). lox2272 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." protein_bind 6195..6228 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 6270..6858 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(6323..6343) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(6741..6752) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 6901..7022 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6912..6931) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 6966..6985 /label=SV40pA-R /note="SV40 polyA, reverse primer" repeat_region 7184..7313 /label=AAV2 ITR ORIGIN 1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt 2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag 2641 gaagatcgta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 2701 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 2761 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 2821 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 2881 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc 2941 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat 3001 gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg 3061 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc 3121 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg 3181 agtcgctgcg acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc 3241 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc 3301 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa 3361 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ctgtccgcgg 3421 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc 3481 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg 3541 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgga tccccataac 3601 ttcgtataaa gtatcctata cgaagttata tcaaaatagg aagaccaatg cttcaccatc 3661 gacccgaatt gccaagcatc accatcgacc cataacttcg tataatgtat gctatacgaa 3721 gttatactag tgctagagct agctgatcac ttaaggttta aacaccggtt catcatttgt 3781 acaattcgtc cattcccagg gtgattcccg ccgccgtcac gaattccagc aacaccatgt 3841 gatctctctt ttcgttcggg tcctttgaca gggccgactg agttgacagg taatgattgt 3901 caggaagcag cacaggacca tctcctatcg gagtattttg ctgatagtgg tctgccagct 3961 ggactgaacc atcttcaata ttgtgcctga ttttgaagtt gaccttgata ccgttcttct 4021 gcttgtctgc catgatgtag acgttatgag agttgtagtt gtactccagc ttgtgaccga 4081 gaatgtttcc atcttcctta aagtcaattc ccttaagttc gatccggttc acgagcgtgt 4141 ctccttcgaa cttgacttcg gcccgagttt tgtagtttcc atcatccttg aaaaagatag 4201 ttctttcctg cacatatcct tcaggcattg cggacttaaa gaaatcatgt tgcttcatat 4261 ggtcagggta ccgcgagaag cattgcactc cgtaggtaag ggtagtcaca agtgtcggcc 4321 acggcactgg cagctttccg gtggtgcaga tgaacttgag agtaagcttt ccgtaggtag 4381 catctccctc tccttctccg gacacggaga atttgtgacc gttcacgtcc ccatccagct 4441 ccaccagaat cggcaccact ccagtgaaga gttcctctcc cttggacaca ggacccgggt 4501 tttcctccac gtcgccgcaa gtcagcagtg acccgcggcc ttctccgcta cctttgtaca 4561 gttcgtccat ccccagggta ataccagccg cagtgacaaa ttccaggagc accatatgat 4621 cccgcttttc gtttgggtcc ttcgacagag cggactgagt ggacaagtag tggttatcag 4681 gcaggagcac cggcccatct ccaatcgggg tattctgttg gtagtgatcg gccaattgca 4741 ccgatccgtc ctcaatgtta tgcctgatct tgaaattgac cttaatacca ttcttctgtt 4801 tgtccgccat aatgtacacg ttgtgggagt tgtaattgta ttcgagctta tgccccaaga 4861 tgttgccatc ttccttgaag tcaataccct tcagttcaat tctgttcacg agggtatccc 4921 cctcaaactt gacctcggcc ctggtcttat agtttccgtc gtccttaaag aagattgtcc 4981 gctcctgcac gtatccttcc ggcatcgcgg atttgaagaa gtcgtgctgc ttcatgtggt 5041 ccgggtaccg ggagaaacat tgcactccgt aagtcaaggt ggtgacaagg gttggccacg 5101 ggactgggag ctttccagtg gtacagataa acttcagagt cagctttccg taagtagcgt 5161 ccccttctcc ttccccggac accgagaact tatgtccgtt cacatctccg tcgagttcga 5221 cgaggatagg caccactccc gtgaacagtt cctccccttt cgacacgggt ccggggttct 5281 cttccacgtc cccggcctgc ttgagcaggg agaagttggt ggcgccggag cccttgtaca 5341 gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat 5401 cgcgcttctc gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg 5461 gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca 5521 cgctgccgtc ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct 5581 tgtcggccat gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga 5641 tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc 5701 cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc 5761 gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt 5821 cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg 5881 gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat 5941 cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga 6001 ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg gtctcgagtc 6061 gcgaacgcgt ggcgcgccgg taccactagt ataacttcgt ataggatact ttatacgaag 6121 ttatcattgg gattcttcct attttgatcc aagcatcacc atcgaccctc tagtccagat 6181 ctcaccatcg acccataact tcgtatagca tacattatac gaagttatgt ccctcgaaga 6241 ggttcgaatt cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa 6301 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa 6361 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat 6421 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt 6481 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc 6541 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc 6601 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg 6661 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga 6721 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc 6781 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc 6841 tttgggccgc ctccccgcat cgataccgtc gacccgggcg gccgcttcga gcagacatga 6901 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 6961 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 7021 ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt 7081 tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga taaggatctt cctagagcat 7141 ggctacgtag ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg 7201 gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc 7261 gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcatt 7321 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgct //