LOCUS       sequence-233764-        7368 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent GFP expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
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  JOURNAL   Unpublished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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-CAG-Flex-Rev-3xGFP
FEATURES             Location/Qualifiers
     source          1..7368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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(join(941..1732,1733..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    3596..3629
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3692..3725
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(3776..4486,4487..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(join(4553..5263,5264..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(join(5333..6043,6044..6046,6047..6049))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(5333..6043,6044..6046,6047..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"
     protein_bind    complement(6091..6124)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(6195..6228)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     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
//
