LOCUS       sequence-350684-        6794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Viral vector for co-expression of non-functional Kir2.1 and EGFP in 
            cells expressing Cre AND NOT Flp driven by the human Synapsin 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6794)
  AUTHORS   Ubina T, Vahedi-Hunter T, Agnew-Svoboda W, Wong W, Gupta A, 
            Santhakumar V, Riccomagno MM
  TITLE     ExBoX - a simple Boolean exclusion strategy to drive expression in 
            neurons.
  JOURNAL   J Cell Sci. 2021 Oct 15;134(20). pii: 272538. doi: 
            10.1242/jcs.257212. Epub 2021 Oct 20.
  PUBMED    34515305
REFERENCE   2  (bases 1 to 6794)
  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: "J Cell 
            Sci."; date: "2021-10-15"; volume: "134(20). pii: 272538. doi"; 
            pages: " 10.1242/jcs.257212. Epub 2021 Oct 20"
COMMENT     Sequence Label: pAAV-SynI-CreOn-FlpOff-Kir2.1MUT-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..6794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    69..89
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        111..558
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    558..582
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(589..622)
                     /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(676..709)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    716..749
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(join(793..1506,1507..1509,1510..1512))
                     /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(796..1506,1507..1509,1510..1512))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(838..859)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1187..1206
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1446..1467
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1513..1569)
                     /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(2869..2898)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      2898..2907
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2944..2977
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    2987..3020
                     /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    3071..3104
                     /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(3105..3129)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3139..3727
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3192..3212)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3610..3621)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3757..3774)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3763..3970
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3978..4118
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3978..4107
                     /label=AAV2 ITR
     rep_origin      4193..4648
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4280..4299)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4490..4511
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4665..4684)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4784..4806
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4844..4862)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4930..5034
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5035..5103,5104..5895)
                     /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(5253..5272)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6066..6654
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6555..6574
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6716..6794,1..62)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(6727..6794,1..62)
                     /label=AAV2 ITR
ORIGIN
        1 ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc
       61 cttctagaca actttgtata gaaaagttgc tgcagagggc cctgcgtatg agtgcaagtg
      121 ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga ccccgaccca
      181 ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga gagggggagg
      241 ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct
      301 tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca
      361 ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc
      421 ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg
      481 cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg
      541 tcgtgcctga gagcgcagca agtttgtaca aaaaagcagg ctgccaccat aacttcgtat
      601 aggatacttt atacgaagtt atccggcaga atggtagctg gattgtagct gctattagca
      661 atatgaaacc tcttaataac ttcgtatagc atacattata cgaagttatg gcgcggaagt
      721 tcctattctc tagaaagtat aggaacttcc ctcgagtacc ggtcacgcgt acgcaagcgc
      781 cttagcggcc gcttacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac
      841 gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg
      901 ggtgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg
      961 ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt
     1021 caccttgatg ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt
     1081 gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc
     1141 gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc
     1201 gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa
     1261 gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag
     1321 ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag
     1381 ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc
     1441 gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc
     1501 cttgctcacc ataggcccgg ggttttcttc aacatctcct gcttgcttta acagagagaa
     1561 gttcgtggct ccgcttcctc cactgcctat ctccgattct cgccttaagg gcctgggctc
     1621 tagaggtacg cttgcctggt tgtggagatc tatgccagga ggtgagtctg tgcttgtgct
     1681 ctctgggact ccgttctcac tatcctcctc ttcctctttg cttgttaggg caacttcatt
     1741 ttcatagcaa aatgaatttg catttgaaag gatgtatttc ttctctgcta agtctctggc
     1801 actacaaagg ggggtgttag gtacttcata agtcttatgg aatcttgaat agtctacttt
     1861 atagtagtgt ttctcttcaa agagcactgg ctcatagcgg tgaccccaga gaatttcatt
     1921 ggccagatac gaactccggc attgagttgt catggcagtc gcctccacca tgccttccag
     1981 tatgacaaca atttcaaagt ctgcattgtc aatgtcctgc ttactcaagt catataaagg
     2041 gctgtcttca tctatttcgt gaacgatagt gatgggggac actagaaata tgcggtcaat
     2101 tccactatca aaaccaacat tgatgtctat ctggtccaaa gggataaact ccccttctga
     2161 agtgatccta gatttgagaa gctgtgcccg gacatgagct cccacaaggt ggctctttcg
     2221 aaggttaccc actctccaca tcaagcagag tttgccatcc ctcatggcaa tcacagcatt
     2281 gtgactgaag acaagagtct catttctctt ctttggcttt gccatcttcg ccatgactgc
     2341 accaatgatg aaggcgtcaa tgatgcagcc tacaattgac tggaatacca ccatgaagac
     2401 agcaattggg cattcgtctg tcacacacct gaaagcagcg gcaatggttg tctgggtctc
     2461 gatggagaag aggaaggcag ccgtgaagct gttaacctcc gacacgcatg ctttgctcac
     2521 tttagaagta tctagatccc catggagcag ggctatcaac caaaacacac agccaaagaa
     2581 cagccaggag agcacgaagg caagacagaa gataaccagc atccacctcc agcggatgtc
     2641 gacacaggta gtaaagatgt ctgccaggta cctctgtccc ttctcaccca cgttgataaa
     2701 ctgaacattg caatgaccat ctttcttcac aaagcggctc ctgcactgtt gtcgggtatg
     2761 gactttactc ttgccattcc caaagccatt ggcaactgcc atagtggcca gcttcatgcc
     2821 atcttcctcc gaagagacaa tgctgtaccg gttggttctc acactgccca ggtcctcctc
     2881 gctgatcagc ttctgctcca tggtggcgga tcctaggtac ctgagctcaa cgatagcact
     2941 agtgaagttc ctattctcta gaaagtatag gaacttcgct agccggataa cttcgtataa
     3001 agtatcctat acgaagttat ttgccttaac ccagaaatta tcactgttat tctttagaat
     3061 ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag ttatacccag ctttcttgta
     3121 caaagtggga attccgataa tcaacctctg gattacaaaa tttgtgaaag attgactggt
     3181 attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat
     3241 catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg
     3301 tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt
     3361 gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact
     3421 ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc
     3481 tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg
     3541 tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc
     3601 tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg
     3661 cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc
     3721 tccccgcatc gggaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     3781 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3841 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3901 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc
     3961 atgctgggga gggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4021 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4081 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     4141 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     4201 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4261 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4321 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4381 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     4441 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4501 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     4561 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4621 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     4681 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4741 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4801 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     4861 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     4921 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     4981 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5041 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5101 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5161 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5221 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5281 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5341 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5401 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5461 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     5521 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     5581 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     5641 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     5701 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
     5761 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     5821 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     5881 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     5941 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6001 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6061 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6121 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6181 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6241 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6301 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6361 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6421 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6481 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6541 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6601 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6661 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgtcctgc
     6721 aggcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg
     6781 acctttggtc gccc
//
