LOCUS       sequence-342036-        6228 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Viral vector for co-expression of NaChBac 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 6228)
  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 6228)
  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-mNaChBac-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..6228
                     /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
     protein_bind    148..168
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        190..637
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    637..661
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(668..701)
                     /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(752..785)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    792..825
                     /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(831..1544,1545..1547,1548..1550))
                     /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(834..1544,1545..1547,1548..1550))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(876..897)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1225..1244
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1484..1505
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1551..1607)
                     /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"
     protein_bind    2460..2493
                     /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    2500..2533
                     /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    2584..2617
                     /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(2618..2642)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2652..3240
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2705..2725)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3123..3134)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3270..3287)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3276..3483
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3491..3631
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3491..3620
                     /label=AAV2 ITR
     rep_origin      3706..4161
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3793..3812)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4003..4024
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4178..4197)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4297..4319
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4357..4375)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4443..4547
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4548..4616,4617..5408)
                     /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(4766..4785)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5579..6167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6068..6087
                     /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 ttctagacaa ctttgtatag aaaagttgct gcagagggcc
      181 ctgcgtatga gtgcaagtgg gttttaggac caggatgagg cggggtgggg gtgcctacct
      241 gacgaccgac cccgacccac tggacaagca cccaaccccc attccccaaa ttgcgcatcc
      301 cctatcagag agggggaggg gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc
      361 agcaccgcgg acagtgcctt cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac
      421 tgaaggcgcg ctgacgtcac tcgccggtcc cccgcaaact ccccttcccg gccaccttgg
      481 tcgcgtccgc gccgccgccg gcccagccgg accgcaccac gcgaggcgcg agataggggg
      541 gcacgggcgc gaccatctgc gctgcggcgc cggcgactca gcgctgcctc agtctgcggt
      601 gggcagcgga ggagtcgtgt cgtgcctgag agcgcagcaa gtttgtacaa aaaagcaggc
      661 tgccaccata acttcgtata ggatacttta tacgaagtta tgcagaatgg tagctggatt
      721 gtagctgcta ttagcaatat gaaacctctt aataacttcg tatagcatac attatacgaa
      781 gttatggcgc ggaagttcct attctctaga aagtatagga acttccctta ttacttgtac
      841 agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag gaccatgtga
      901 tcgcgcttct cgttggggtc tttgctcagg gcggactggg tgctcaggta gtggttgtcg
      961 ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc
     1021 acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc gttcttctgc
     1081 ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt gtgccccagg
     1141 atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg
     1201 ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg
     1261 cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg
     1321 tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag ggtgggccag
     1381 ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca
     1441 tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg
     1501 accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat aggcccgggg
     1561 ttttcttcaa catctcctgc ttgctttaac agagagaagt tcgtggctcc gcttcctcca
     1621 ctgccgatat ccttaagttg tttgagcaag ctcttcagtt cggctacatc ctttcgcaat
     1681 gctgaaatct cctgtttaag tccgtcggcc tccccatctt cctcattgtc cgtaagctcc
     1741 gccttttcca cgttgttcac aatgactccg atgaaaagat tgaaaatgat aaaagtacca
     1801 atcaggacaa acgagacaaa gtagagccat gaccagggca cctctgcaaa aatgggccgc
     1861 attacaccgc tggcccagct ctccaaggtc accacttgga acagcgtcag caacgaaagc
     1921 tggaggttcc caaagtattc aggcgatacg tgctggaaga gcattgtccc gatcactgca
     1981 aagatataga agaagatgga catcaagatc aagatattgc ccaacgccgg aatcgtcatc
     2041 accagcgcat ctacgagtct gcgcagggag gggacgacgg agatcgccct gagtacccgc
     2101 agcactctaa ggattcgcaa tactgtcacg aactgggctc ccgcgaagat atgcccagcc
     2161 gcgacaatca gaaaatcaaa ccaattccac gagctgcgga agaaggccga ctttgggttc
     2221 gacgccaaaa acctcatcgc gatctcgatg gtgaagatcc aaagcaggac gaggtcaatc
     2281 cgataaaaga gccacttgtg gtccgcgtag atcctcggat atgtttcgat tcccacgatc
     2341 agtgcattaa agaggatcag ggcaatgacg gtgaaagtaa acgctctatg gttgacaatt
     2401 ttctgcatct ttgacgtgaa tgagttctgt ttctggcgtg cttccatttt catggtggcg
     2461 aagttcctat tctctagaaa gtataggaac ttcgctagca taacttcgta taaagtatcc
     2521 tatacgaagt tatttgcctt aacccagaaa ttatcactgt tattctttag aatggtgcaa
     2581 agaataactt cgtataatgt atgctatacg aagttatacc cagctttctt gtacaaagtg
     2641 ggaattccga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     2701 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     2761 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     2821 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     2881 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     2941 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3001 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc
     3061 catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     3121 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     3181 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     3241 atcgggaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     3301 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3361 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3421 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg
     3481 ggagggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     3541 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     3601 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     3661 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     3721 gcgcattaag cgcggcgggg gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3781 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3841 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3901 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     3961 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4021 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     4081 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4141 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     4201 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4261 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4321 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     4381 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     4441 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4501 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4561 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4621 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4681 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4741 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4801 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4861 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4921 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4981 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5041 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5101 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5161 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5221 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
     5281 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5341 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5401 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5461 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5521 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5581 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5641 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5701 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     5761 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5821 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5881 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5941 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6001 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6061 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6121 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6181 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
