LOCUS       sequence-350660-        6308 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 6308)
  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 6308)
  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..6308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      149..604
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(236..255)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     446..467
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(621..640)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     740..762
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(800..818)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        886..990
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(991..1059,1060..1851)
                     /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(1209..1228)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2022..2610
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2511..2530
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2672..2812
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2683..2812
                     /label=AAV2 ITR
     protein_bind    2819..2839
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        2861..3308
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    3308..3332
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3339..3372)
                     /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(3426..3459)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    3466..3499
                     /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(3543..4256,4257..4259,4260..4262))
                     /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(3546..4256,4257..4259,4260..4262))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3588..3609)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3937..3956
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4196..4217
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(4263..4319)
                     /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    5208..5241
                     /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    5251..5284
                     /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    5335..5368
                     /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(5369..5393)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    5403..5991
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5456..5476)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5874..5885)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6021..6038)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6027..6234
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   join(6242..6308,1..74)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(6242..6308,1..63)
                     /label=AAV2 ITR
ORIGIN
        1 accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg
       61 cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
      121 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg
      181 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct
      241 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
      301 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
      361 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
      421 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
      481 tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta
      541 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca
      601 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
      661 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
      721 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
      781 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
      841 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
      901 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
      961 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     1021 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     1081 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     1141 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     1201 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     1261 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     1321 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     1381 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     1441 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     1501 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     1561 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     1621 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     1681 gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact ggggccagat
     1741 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     1801 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     1861 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     1921 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     1981 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2041 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2101 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2161 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2221 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2281 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2341 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2401 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2461 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     2521 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2581 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2641 ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc tcgctcgctc
     2701 actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
     2761 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc cttctagaca
     2821 actttgtata gaaaagttgc tgcagagggc cctgcgtatg agtgcaagtg ggttttagga
     2881 ccaggatgag gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc
     2941 acccaacccc cattccccaa attgcgcatc ccctatcaga gagggggagg ggaaacagga
     3001 tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc
     3061 ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc
     3121 ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg
     3181 gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg cgctgcggcg
     3241 ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga
     3301 gagcgcagca agtttgtaca aaaaagcagg ctgccaccat aacttcgtat aggatacttt
     3361 atacgaagtt atccggcaga atggtagctg gattgtagct gctattagca atatgaaacc
     3421 tcttaataac ttcgtatagc atacattata cgaagttatg gcgcggaagt tcctattctc
     3481 tagaaagtat aggaacttcc ctcgagtacc ggtcacgcgt acgcaagcgc cttagcggcc
     3541 gcttacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc
     3601 aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg
     3661 tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg
     3721 tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg
     3781 ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc
     3841 ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc
     3901 accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg
     3961 aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc
     4021 tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg
     4081 agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg
     4141 ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg
     4201 ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc
     4261 ataggcccgg ggttttcttc aacatctcct gcttgcttta acagagagaa gttcgtggct
     4321 ccgcttcctc cactgccgat atccttaagt tgtttgagca agctcttcag ttcggctaca
     4381 tcctttcgca atgctgaaat ctcctgttta agtccgtcgg cctccccatc ttcctcattg
     4441 tccgtaagct ccgccttttc cacgttgttc acaatgactc cgatgaaaag attgaaaatg
     4501 ataaaagtac caatcaggac aaacgagaca aagtagagcc atgaccaggg cacctctgca
     4561 aaaatgggcc gcattacacc gctggcccag ctctccaagg tcaccacttg gaacagcgtc
     4621 agcaacgaaa gctggaggtt cccaaagtat tcaggcgata cgtgctggaa gagcattgtc
     4681 ccgatcactg caaagatata gaagaagatg gacatcaaga tcaagatatt gcccaacgcc
     4741 ggaatcgtca tcaccagcgc atctacgagt ctgcgcaggg aggggacgac ggagatcgcc
     4801 ctgagtaccc gcagcactct aaggattcgc aatactgtca cgaactgggc tcccgcgaag
     4861 atatgcccag ccgcgacaat cagaaaatca aaccaattcc acgagctgcg gaagaaggcc
     4921 gactttgggt tcgacgccaa aaacctcatc gcgatctcga tggtgaagat ccaaagcagg
     4981 acgaggtcaa tccgataaaa gagccacttg tggtccgcgt agatcctcgg atatgtttcg
     5041 attcccacga tcagtgcatt aaagaggatc agggcaatga cggtgaaagt aaacgctcta
     5101 tggttgacaa ttttctgcat ctttgacgtg aatgagttct gtttctggcg tgcttccatt
     5161 ttcatggtgg cggatcctag gtacctgagc tcaacgatag cactagtgaa gttcctattc
     5221 tctagaaagt ataggaactt cgctagccgg ataacttcgt ataaagtatc ctatacgaag
     5281 ttatttgcct taacccagaa attatcactg ttattcttta gaatggtgca aagaataact
     5341 tcgtataatg tatgctatac gaagttatac ccagctttct tgtacaaagt gggaattccg
     5401 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     5461 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     5521 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     5581 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     5641 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     5701 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     5761 tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt ccatggctgc
     5821 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     5881 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     5941 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgggaat
     6001 tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     6061 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     6121 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     6181 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagggccg
     6241 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     6301 gccgggcg
//
