LOCUS       sequence-450290-        5767 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent AAV-mediated expression of GFP under the CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5767)
  TITLE     Accessory plasmids for optogenetics
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5767)
  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            "Unpublished"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-FLEX-GFP
FEATURES             Location/Qualifiers
     source          1..5767
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   826..955
                     /label=AAV2 ITR
     polyA_signal    1117..1238
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1154..1173)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1208..1227
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    1281..1869
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1387..1398
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1796..1816
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    1911..1944
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2015..2048
                     /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)."
     regulatory      2065..2074
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2071..2073,2074..2076,2077..2790)
                     /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             join(2071..2073,2074..2076,2077..2787)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2116..2137)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2377..2396)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2724..2745
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2813..2846)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(2909..2942)
                     /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)."
     primer_bind     complement(2977..2996)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(3058..3080)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(3359..3639)
                     /label=chicken beta-actin promoter
     repeat_region   3950..4079
                     /label=AAV2 ITR
     rep_origin      4151..4606
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4238..4257)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4448..4469
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4632..4736
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4737..4805,4806..5597)
                     /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(4955..4974)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg cgctcgctcg
      841 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca
      901 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag
      961 ttaatgatta acccgccatg ctacttatct acgtagccat gctctaggaa gatccttatc
     1021 gattttacca catttgtaga ggttttactt gctttaaaaa acctcccaca tctccccctg
     1081 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     1141 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     1201 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgctc gaagcggccg
     1261 cccgggtcga cggtatcgat gcggggaggc ggcccaaagg gagatccgac tcgtctgagg
     1321 gcgaaggcga agacgcggaa gaggccgcag agccggcagc aggccgcggg aaggaaggtc
     1381 cgctggattg agggccgaag ggacgtagca gaaggacgtc ccgcgcagaa tccaggtggc
     1441 aacacaggcg agcagccaag gaaaggacga tgatttcccc gacaacacca cggaattgtc
     1501 agtgcccaac agccgagccc ctgtccagca gcgggcaagg caggcggcga tgagttccgc
     1561 cgtggcaata gggaggggga aagcgaaagt cccggaaagg agctgacagg tggtggcaat
     1621 gccccaacca gtgggggttg cgtcagcaaa cacagtgcac accacgccac gttgcctgac
     1681 aacgggccac aactcctcat aaagagacag caaccaggat ttatacaagg aggagaaaat
     1741 gaaagccata cgggaagcaa tagcatgata caaaggcatt aaagcagcgt atccacatag
     1801 cgtaaaagga gcaacatagt taagaatacc agtcaatctt tcacaaattt tgtaatccag
     1861 aggttgatta tcgataagct tgatatcgaa ttcgaacctc ttcgagggac ataacttcgt
     1921 ataatgtatg ctatacgaag ttatgggtcg atggtgagat ctggactaga gggtcgatgg
     1981 tgatgcttgg atcaaaatag gaagaatccc aatgataact tcgtataaag tatcctatac
     2041 gaagttatac tagaattcgg taccgccacc atggtgagca agggcgagga gctgttcacc
     2101 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2161 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catttgcacc
     2221 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     2281 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2341 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2401 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2461 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     2521 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     2581 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     2641 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     2701 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2761 actctcggca tggacgagct gtacaagtaa aactcgaggg gggatcccta gtataacttc
     2821 gtatagcata cattatacga agttatgggt cgatggtgat gcttggcaat tcgggtcgat
     2881 ggtgaagcat tggtcttcct attttgatat aacttcgtat aggatacttt atacgaagtt
     2941 atggggatcc aattctttgc caaaatgatg agacagcaca ataaccagca cgttgcccag
     3001 gagctgtagg aaaaagaaga aggcatgaac atggttagca gaggctctag agccgccggt
     3061 cacacgccag aagccgaacc ccgccctgcc ccgtcccccc cgaaggcagc cgtccccccg
     3121 cggacagccc cgagccgctc cccccgcaca aagggccctc ccggagcccc tcaaggcttt
     3181 cacgcagcca cagaaaagaa acaagccgtc attaaaccaa gcgctaatta cagcccggag
     3241 gagaagggcc gtcccgcccg ctcacctgtg ggagtaacgc ggtcagtcag agccggggcg
     3301 ggcggcgcga ggcggcggcg gagcggggca cggggcgaag gcagcgcgca gcgactcccg
     3361 cccgccgcgc gcttcgcttt ttatagggcc gccgccgccg ccgcctcgcc ataaaaggaa
     3421 actttcggag cgcgccgctc tgattggctg ccgccgcacc tctccgcctc gccccgcccc
     3481 gcccctcgcc ccgccccgcc ccgcctggcg cgcgcccccc cccccccccc cccgccccca
     3541 tcgctgcaca aaataattaa aaaataaata aatacaaaat tgggggtggg gagggggggg
     3601 agatggggag agtgaagcag aacgtggggc tcacctcgac catggtaata gcgatgacta
     3661 atacgtagat gtactgccaa gtaggaaagt cccataaggt catgtactgg gcataatgcc
     3721 aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt acttggcata tgatacactt
     3781 gatgtactgc caagtgggca gtttaccgta aatactccac ccattgacgt caatggaaag
     3841 tccctattgg cgttactatg ggaacatacg tcattattga cgtcaatggt actcttccta
     3901 gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta
     3961 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     4021 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     4081 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     4141 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4201 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4261 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4321 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     4381 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4441 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     4501 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     4561 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     4621 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4681 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     4741 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     4801 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     4861 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     4921 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     4981 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5041 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5101 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5161 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5221 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     5281 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     5341 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     5401 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     5461 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     5521 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     5581 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     5641 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5701 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5761 gatcttc
//
