LOCUS       sequence-9543-ex        6324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-activated AAV expression of EYFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6324)
  TITLE     Cre-activated AAV expression plasmids
REFERENCE   2  (bases 1 to 6324)
  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"
COMMENT     Sequence Label: pAAV-EF1a-double floxed-EYFP-WPRE-HGHpA
FEATURES             Location/Qualifiers
     source          1..6324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1450..1483)
                     /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(1534..1567)
                     /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(1579..2292,2293..2295,2296..2298))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1624..1645)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1973..1992
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2232..2253
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2295..2304
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2309..2342
                     /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    2393..2426
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2451..3039
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2504..2524)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2922..2933)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3071..3547
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3270..3289)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3587..3727
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3587..3716
                     /label=AAV2 ITR
     rep_origin      3802..4257
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3889..3908)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4099..4120
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4274..4293)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4393..4415
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4453..4471)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4539..4643
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4644..4712,4713..5504)
                     /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(4862..4881)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5675..6263
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6164..6183
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1441 gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg
     1561 aagttatggc gcgccctatt acttgtacag ctcgtccatg ccgagagtga tcccggcggc
     1621 ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagggc
     1681 ggactggtag ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt
     1741 gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt ggcggatctt
     1801 gaagttcacc ttgatgccgt tcttctgctt gtcggccatg atatagacgt tgtggctgtt
     1861 gtagttgtac tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt
     1921 cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta
     1981 gttgccgtcg tccttgaaga agatggtgcg ctcctggacg tagccttcgg gcatggcgga
     2041 cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg gcgaagcact gcaggccgta
     2101 gccgaaggtg gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa
     2161 cttcagggtc agcttgccgt aggtggcatc gccctcgccc tcgccggaca cgctgaactt
     2221 gtggccgttt acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc
     2281 ctcgcccttg ctcaccatgg tggctagcat aacttcgtat aaagtatcct atacgaagtt
     2341 atttgcctta acccagaaat tatcactgtt attctttaga atggtgcaaa gaataacttc
     2401 gtataatgta tgctatacga agttatgaat tcgatatcaa gcttatcgat aatcaacctc
     2461 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     2521 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     2581 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     2641 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     2701 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     2761 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     2821 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctatgttg
     2881 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     2941 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     3001 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccga gcgctgctcg
     3061 agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt
     3121 tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga
     3181 ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt
     3241 tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc
     3301 acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc
     3361 tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg
     3421 gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat
     3481 ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc
     3541 tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg
     3601 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     3661 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     3721 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3781 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3841 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3901 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3961 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     4021 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4081 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     4141 cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4201 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     4261 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     4321 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     4381 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     4441 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     4501 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4561 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     4621 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     4681 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     4741 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     4801 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     4861 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     4921 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     4981 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5041 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5101 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     5161 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     5221 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     5281 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     5341 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     5401 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     5461 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     5521 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5581 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5641 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5701 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5761 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5821 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5881 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5941 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6001 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6061 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6121 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6181 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6241 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6301 tttgctggcc ttttgctcac atgt
//
