LOCUS       sequence-237802-        7365 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 7365)
  TITLE     AAV expression plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7365)
  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            "Unpublished"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..7365
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..278
                     /label=chicken beta-actin promoter
     primer_bind     557..579
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     641..660
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    695..728
                     /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    791..824
                     /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(875..1585,1586..1588))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(1589..1642)
                     /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(1652..2362,2363..2365))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(2366..2422)
                     /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(2432..3142,3143..3145,3146..3148))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2432..3142,3143..3145,3146..3148))
                     /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(3190..3223)
                     /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(3294..3327)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3369..3957
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3422..3442)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3840..3851)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4000..4121
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4011..4030)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4065..4084
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4283..4412
                     /label=AAV2 ITR
     rep_origin      complement(4649..5237)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4729..4748)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5408..6199,6200..6268))
                     /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     6031..6050
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6269..6373)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6399..6854)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6536..6557)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6748..6767
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6926..7055
                     /label=AAV2 ITR
ORIGIN
        1 tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa
       61 ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg
      121 ggcgcgcgcc rggsggggsg gggsggggsg rggggsgggg sggggsgagg cggagaggtg
      181 cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
      241 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
      301 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
      361 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg
      421 tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg
      481 gccctttgtg cggggggagc ggctcggggc tgtccgcggg gggacggctg ccttcggggg
      541 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
      601 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg
      661 ctgtctcatc attttggcaa agaattggat ccccataact tcgtataaag tatcctatac
      721 gaagttatat caaaatagga agaccaatgc ttcaccatcg acccgaattg ccaagcatca
      781 ccatcgaccc ataacttcgt ataatgtatg ctatacgaag ttatactagt gctagagcta
      841 gctgatcact taaggtttaa acaccggttc atcatttgta caattcgtcc attcccaggg
      901 tgattcccgc cgccgtcacg aattccagca acaccatgtg atctctcttt tcgttcgggt
      961 cctttgacag ggccgactga gttgacaggt aatgattgtc aggaagcagc acaggaccat
     1021 ctcctatcgg agtattttgc tgatagtggt ctgccagctg gactgaacca tcttcaatat
     1081 tgtgcctgat tttgaagttg accttgatac cgttcttctg cttgtctgcc atgatgtaga
     1141 cgttatgaga gttgtagttg tactccagct tgtgaccgag aatgtttcca tcttccttaa
     1201 agtcaattcc cttaagttcg atccggttca cgagcgtgtc tccttcgaac ttgacttcgg
     1261 cccgagtttt gtagtttcca tcatccttga aaaagatagt tctttcctgc acatatcctt
     1321 caggcattgc ggacttaaag aaatcatgtt gcttcatatg gtcagggtac cgcgagaagc
     1381 attgcactcc gtaggtaagg gtagtcacaa gtgtcggcca cggcactggc agctttccgg
     1441 tggtgcagat gaacttgaga gtaagctttc cgtaggtagc atctccctct ccttctccgg
     1501 acacggagaa tttgtgaccg ttcacgtccc catccagctc caccagaatc ggcaccactc
     1561 cagtgaagag ttcctctccc ttggacacag gacccgggtt ttcctccacg tcgccgcaag
     1621 tcagcagtga cccgcggcct tctccgctac ctttgtacag ttcgtccatc cccagggtaa
     1681 taccagccgc agtgacaaat tccaggagca ccatatgatc ccgcttttcg tttgggtcct
     1741 tcgacagagc ggactgagtg gacaagtagt ggttatcagg caggagcacc ggcccatctc
     1801 caatcggggt attctgttgg tagtgatcgg ccaattgcac cgatccgtcc tcaatgttat
     1861 gcctgatctt gaaattgacc ttaataccat tcttctgttt gtccgccata atgtacacgt
     1921 tgtgggagtt gtaattgtat tcgagcttat gccccaagat gttgccatct tccttgaagt
     1981 caataccctt cagttcaatt ctgttcacga gggtatcccc ctcaaacttg acctcggccc
     2041 tggtcttata gtttccgtcg tccttaaaga agattgtccg ctcctgcacg tatccttccg
     2101 gcatcgcgga tttgaagaag tcgtgctgct tcatgtggtc cgggtaccgg gagaaacatt
     2161 gcactccgta agtcaaggtg gtgacaaggg ttggccacgg gactgggagc tttccagtgg
     2221 tacagataaa cttcagagtc agctttccgt aagtagcgtc cccttctcct tccccggaca
     2281 ccgagaactt atgtccgttc acatctccgt cgagttcgac gaggataggc accactcccg
     2341 tgaacagttc ctcccctttc gacacgggtc cggggttctc ttccacgtcc ccggcctgct
     2401 tgagcaggga gaagttggtg gcgccggagc ccttgtacag ctcgtccatg ccgagagtga
     2461 tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg ttggggtctt
     2521 tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc
     2581 cgatgggggt gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt
     2641 ggcggatctt gaagttcacc ttgatgccgt tcttctgctt gtcggccatg atatagacgt
     2701 tgtggctgtt gtagttgtac tccagcttgt gccccaggat gttgccgtcc tccttgaagt
     2761 cgatgccctt cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc
     2821 gggtcttgta gttgccgtcg tccttgaaga agatggtgcg ctcctggacg tagccttcgg
     2881 gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact
     2941 gcacgccgta ggtcagggtg gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg
     3001 tgcagatgaa cttcagggtc agcttgccgt aggtggcatc gccctcgccc tcgccggaca
     3061 cgctgaactt gtggccgttt acgtcgccgt ccagctcgac caggatgggc accaccccgg
     3121 tgaacagctc ctcgcccttg ctcaccatgg tctcgagtcg cgaacgcgtg gcgcgccggt
     3181 accactagta taacttcgta taggatactt tatacgaagt tatcattggg attcttccta
     3241 ttttgatcca agcatcacca tcgaccctct agtccagatc tcaccatcga cccataactt
     3301 cgtatagcat acattatacg aagttatgtc cctcgaagag gttcgaattc gatatcaagc
     3361 ttatcgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3421 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3481 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3541 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3601 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3661 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3721 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     3781 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3841 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3901 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc
     3961 gataccgtcg acccgggcgg ccgcttcgag cagacatgat aagatacatt gatgagtttg
     4021 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
     4081 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
     4141 attttatgtt tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct
     4201 acaaatgtgg taaaatcgat aaggatcttc ctagagcatg gctacgtaga taagtagcat
     4261 ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg
     4321 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     4381 cgggcggcct cagtgagcga gcgagcgcgc agctgcatta atgaatcggc caacgcgcgg
     4441 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4501 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4561 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4621 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4681 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4741 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4801 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4861 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4921 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4981 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5041 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5101 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     5161 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     5221 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     5281 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     5341 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5401 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5461 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5521 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5581 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5641 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5701 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5761 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5821 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5881 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5941 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6001 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6061 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6121 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6181 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6241 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     6301 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6361 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     6421 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     6481 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     6541 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     6601 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     6661 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     6721 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     6781 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     6841 gctacagggc gcgtcccatt cgcccttcag gctgcgcaac tgttgggaag ggcgatcggt
     6901 gcgggcctct tcgctattac gccagctgcg cgctcgctcg ctcactgagg ccgcccgggc
     6961 aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag
     7021 agagggagtg gccaactcca tcactagggg ttccttgtag ttaatgatta acccgccatg
     7081 ctacttatct acgtagccat gctctaggaa gagtaccatt gacgtcaata atgacgtatg
     7141 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     7201 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     7261 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     7321 ctacttggca gtacatctac gtattagtca tcgctattac catgg
//
