LOCUS       sequence-350556-        6794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Viral vector for co-expression of 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-Kir2.1-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..6794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    132..152
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        174..621
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    621..645
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(652..685)
                     /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(739..772)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    779..812
                     /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(856..1569,1570..1572,1573..1575))
                     /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(859..1569,1570..1572,1573..1575))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(901..922)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1250..1269
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1509..1530
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1576..1632)
                     /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(2932..2961)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     regulatory      2961..2970
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3007..3040
                     /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    3050..3083
                     /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    3134..3167
                     /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(3168..3192)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3202..3790
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3255..3275)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3673..3684)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3820..3837)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3826..4033
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4041..4181
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4041..4170
                     /label=AAV2 ITR
     rep_origin      4256..4711
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4343..4362)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4553..4574
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4728..4747)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4847..4869
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4907..4925)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4993..5097
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5098..5166,5167..5958)
                     /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(5316..5335)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6129..6717
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6618..6637
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(6779..6794,1..125)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(6790..6794,1..125)
                     /label=AAV2 ITR
ORIGIN
        1 cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg
       61 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg
      121 ttccttctag acaactttgt atagaaaagt tgctgcagag ggccctgcgt atgagtgcaa
      181 gtgggtttta ggaccaggat gaggcggggt gggggtgcct acctgacgac cgaccccgac
      241 ccactggaca agcacccaac ccccattccc caaattgcgc atcccctatc agagaggggg
      301 aggggaaaca ggatgcggcg aggcgcgtgc gcactgccag cttcagcacc gcggacagtg
      361 ccttcgcccc cgcctggcgg cgcgcgccac cgccgcctca gcactgaagg cgcgctgacg
      421 tcactcgccg gtcccccgca aactcccctt cccggccacc ttggtcgcgt ccgcgccgcc
      481 gccggcccag ccggaccgca ccacgcgagg cgcgagatag gggggcacgg gcgcgaccat
      541 ctgcgctgcg gcgccggcga ctcagcgctg cctcagtctg cggtgggcag cggaggagtc
      601 gtgtcgtgcc tgagagcgca gcaagtttgt acaaaaaagc aggctgccac cataacttcg
      661 tataggatac tttatacgaa gttatccggc agaatggtag ctggattgta gctgctatta
      721 gcaatatgaa acctcttaat aacttcgtat agcatacatt atacgaagtt atggcgcgga
      781 agttcctatt ctctagaaag tataggaact tccctcgagt accggtcacg cgtacgcaag
      841 cgccttagcg gccgcttact tgtacagctc gtccatgccg agagtgatcc cggcggcggt
      901 cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga
      961 ctgggtgctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt
     1021 ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa
     1081 gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta
     1141 gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag
     1201 ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt
     1261 gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt
     1321 gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt
     1381 cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt
     1441 cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg
     1501 gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc
     1561 gcccttgctc accataggcc cggggttttc ttcaacatct cctgcttgct ttaacagaga
     1621 gaagttcgtg gctccgcttc ctccactgcc tatctccgat tctcgcctta agggcctggg
     1681 ctctagaggt acgcttgcct ggttgtggag atctatgcca ggaggtgagt ctgtgcttgt
     1741 gctctctggg actccgttct cactatcctc ctcttcctct ttgcttgtta gggcaacttc
     1801 attttcatag caaaatgaat ttgcatttga aaggatgtat ttcttctctg ctaagtctct
     1861 ggcactacaa aggggggtgt taggtacttc ataagtctta tggaatcttg aatagtctac
     1921 tttatagtag tgtttctctt caaagagcac tggctcatag cggtgacccc agagaatttc
     1981 attggccaga tacgaactcc ggcattgagt tgtcatggca gtcgcctcca ccatgccttc
     2041 cagtatgaca acaatttcaa agtctgcatt gtcaatgtcc tgcttactca agtcatataa
     2101 agggctgtct tcatctattt cgtgaacgat agtgatgggg gacactagaa atatgcggtc
     2161 aattccacta tcaaaaccaa cattgatgtc tatctggtcc aaagggataa actccccttc
     2221 tgaagtgatc ctagatttga gaagctgtgc ccggacatga gctcccacaa ggtggctctt
     2281 tcgaaggtta cccactctcc acatcaagca gagtttgcca tccctcatgg caatcacagc
     2341 attgtgactg aagacaagag tctcatttct cttctttggc tttgccatct tcgccatgac
     2401 tgcaccaatg atgaaggcgt caatgatgca gcctacaatt gactggaata ccaccatgaa
     2461 gacagcaatt gggcattcgt ctgtcacaca cctgaaacca tagccaatgg ttgtctgggt
     2521 ctcgatggag aagaggaagg cagccgtgaa gctgttaacc tccgacacgc atgctttgct
     2581 cactttagaa gtatctagat ccccatggag cagggctatc aaccaaaaca cacagccaaa
     2641 gaacagccag gagagcacga aggcaagaca gaagataacc agcatccacc tccagcggat
     2701 gtcgacacag gtagtaaaga tgtctgccag gtacctctgt cccttctcac ccacgttgat
     2761 aaactgaaca ttgcaatgac catctttctt cacaaagcgg ctcctgcact gttgtcgggt
     2821 atggacttta ctcttgccat tcccaaagcc attggcaact gccatagtgg ccagcttcat
     2881 gccatcttcc tccgaagaga caatgctgta ccggttggtt ctcacactgc ccaggtcctc
     2941 ctcgctgatc agcttctgct ccatggtggc ggatcctagg tacctgagct caacgatagc
     3001 actagtgaag ttcctattct ctagaaagta taggaacttc gctagccgga taacttcgta
     3061 taaagtatcc tatacgaagt tatttgcctt aacccagaaa ttatcactgt tattctttag
     3121 aatggtgcaa agaataactt cgtataatgt atgctatacg aagttatacc cagctttctt
     3181 gtacaaagtg ggaattccga taatcaacct ctggattaca aaatttgtga aagattgact
     3241 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3301 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     3361 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     3421 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     3481 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     3541 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg
     3601 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     3661 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     3721 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     3781 gcctccccgc atcgggaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt
     3841 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3901 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3961 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca
     4021 ggcatgctgg ggagggccgc aggaacccct agtgatggag ttggccactc cctctctgcg
     4081 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     4141 ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt
     4201 tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg
     4261 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     4321 cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     4381 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     4441 ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg
     4501 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     4561 ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga tttataaggg
     4621 attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4681 aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct
     4741 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4801 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4861 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     4921 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     4981 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5041 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5101 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5161 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5221 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5281 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5341 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5401 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5461 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5521 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5581 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5641 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5701 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5761 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc
     5821 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5881 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5941 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6001 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6061 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6121 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6181 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6241 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6301 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6361 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6421 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6481 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6541 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6601 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6661 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6721 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc
     6781 tgcaggcagc tgcg
//
