LOCUS       sequence-193613-        5520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV vector that expresses EGFP in a Cre-dependent manner (DIO, 
            double floxed inverted open reading frame)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5520)
  TITLE     Ian Wickersham AAV plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5520)
  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            "Unpublished"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.synP.DIO.EGFP.WPRE.hGH
FEATURES             Location/Qualifiers
     source          1..5520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        150..597
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(624..657)
                     /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(708..741)
                     /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(753..1466,1467..1469,1470..1472))
                     /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             complement(join(756..1466,1467..1469,1470..1472))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(798..819)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1147..1166
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1406..1427
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1469..1478
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1483..1516
                     /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    1567..1600
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1625..2213
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1678..1698)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2096..2107)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2245..2721
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2444..2463)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   2761..2901
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2761..2890
                     /label=AAV2 ITR
     rep_origin      2976..3431
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3063..3082)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3273..3294
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3448..3467)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3567..3589
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3627..3645)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3713..3817
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3818..3886,3887..4678)
                     /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(4036..4055)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4849..5437
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5338..5357
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(5499..5520,1..108)
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 ctcgctcact gaggccgccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
       61 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcacgc
      121 gtgctagact gcagagggcc ctgcgtatga gtgcaagtgg gttttaggac caggatgagg
      181 cggggtgggg gtgcctacct gacgaccgac cccgacccac tggacaagca cccaaccccc
      241 attccccaaa ttgcgcatcc cctatcagag agggggaggg gaaacaggat gcggcgaggc
      301 gcgtgcgcac tgccagcttc agcaccgcgg acagtgcctt cgcccccgcc tggcggcgcg
      361 cgccaccgcc gcctcagcac tgaaggcgcg ctgacgtcac tcgccggtcc cccgcaaact
      421 ccccttcccg gccaccttgg tcgcgtccgc gccgccgccg gcccagccgg accgcaccac
      481 gcgaggcgcg agataggggg gcacgggcgc gaccatctgc gctgcggcgc cggcgactca
      541 gcgctgcctc agtctgcggt gggcagcgga ggagtcgtgt cgtgcctgag agcgcagtcg
      601 aattctctag agtcgactcc ggaataactt cgtataggat actttatacg aagttatgca
      661 gaatggtagc tggattgtag ctgctattag caatatgaaa cctcttaata acttcgtata
      721 gcatacatta tacgaagtta tggcgcgccc tattacttgt acagctcgtc catgccgaga
      781 gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt ctcgttgggg
      841 tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag cacggggccg
      901 tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg
      961 ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc catgatatag
     1021 acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg
     1081 aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg
     1141 gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct
     1201 tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag
     1261 cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg
     1321 gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg
     1381 gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc
     1441 ccggtgaaca gctcctcgcc cttgctcacc atggtggcta gcataacttc gtataaagta
     1501 tcctatacga agttatttgc cttaacccag aaattatcac tgttattctt tagaatggtg
     1561 caaagaataa cttcgtataa tgtatgctat acgaagttat gaattcgata tcaagcttat
     1621 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     1681 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     1741 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     1801 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     1861 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     1921 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     1981 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     2041 gctcgcctat gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     2101 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     2161 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata
     2221 ccgagcgctg ctcgagagat ctacgggtgg catccctgtg acccctcccc agtgcctctc
     2281 ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc
     2341 atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg
     2401 agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc
     2461 tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt
     2521 ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa
     2581 tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc
     2641 taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc
     2701 actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc
     2761 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     2821 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     2881 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
     2941 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     3001 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
     3061 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     3121 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     3181 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     3241 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     3301 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
     3361 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     3421 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3481 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3541 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3601 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     3661 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     3721 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3781 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3841 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3901 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3961 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4021 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4081 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4141 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4201 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4261 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4321 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     4381 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4441 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     4501 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     4561 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     4621 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     4681 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4741 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4801 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4861 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4921 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4981 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5041 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5101 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5161 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5221 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5281 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5341 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5401 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5461 acggttcctg gccttttgct ggccttttgc tcacatgtcc tgcaggcagc tgcgcgctcg
//
