LOCUS       sequence-197190-        7325 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses splitTVA-EGFP-B19G.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7325)
  AUTHORS   Kohara K, Pignatelli M, Rivest AJ, Jung HY, Kitamura T, Suh J, Frank
            D, Kajikawa K, Mise N, Obata Y, Wickersham IR, Tonegawa S
  TITLE     Cell type-specific genetic and optogenetic tools reveal hippocampal 
            CA2 circuits.
  JOURNAL   Nat Neurosci. 2014 Feb;17(2):269-79. doi: 10.1038/nn.3614. Epub 2013
            Dec 15.
  PUBMED    24336151
REFERENCE   2  (bases 1 to 7325)
  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"; doi: "10.1038/nn.3614"; 
            journalName: "Nat Neurosci"; date: "2014-02"; volume: "17"; issue: 
            "2"; pages: "269-79"
COMMENT     Sequence Label: pAAV-synP-FLEX-splitTVA-EGFP-B19G
FEATURES             Location/Qualifiers
     source          1..7325
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      233..242
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    248..281
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    332..365
                     /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)."
     CDS             complement(1946..2002)
                     /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(join(2012..2722,2723..2725,2726..2728))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2012..2722,2723..2725,2726..2728))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2054..2075)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2403..2422
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2662..2683
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2729..2785)
                     /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    complement(3268..3301)
                     /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(3352..3385)
                     /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)."
     misc_feature    3387..3975
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3440..3460)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3858..3869)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3983..4207
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4209..4349
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4209..4338
                     /label=AAV2 ITR
     rep_origin      4424..4879
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4511..4530)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4721..4742
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4896..4915)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5015..5037
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5075..5093)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5161..5265
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5266..5334,5335..6126)
                     /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(5484..5503)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6297..6885
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6786..6805
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6947..7076
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        join(7104..7325,1..226)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
ORIGIN
        1 cctcagcact gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc cccttcccgg
       61 ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg cgaggcgcga
      121 gatagggggg cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag cgctgcctca
      181 gtctgcggtg ggcagcggag gagtcgtgtc gtgcctgaga gcgcagtcag ccgccaccat
      241 ggttaatata acttcgtata atgtatgcta tacgaagtta tgcagaatgg tagctggatt
      301 gtagctgcta ttagcaatat gaaacctctt aataacttcg tataaagtat cctatacgaa
      361 gttatggtac ctacagtctg gtctcacccc cactcttgtg tgattcccat gaagatatga
      421 tcttcccgct ttggggagtg actgacacct ccctccctgt ccctctgaga ttgtgttgcg
      481 taggttctga tcgattgact cttctacaac atgtcatcag gaaaattatc aacatcaagg
      541 cagtcagggc ccctgcactc agtaatacat acttccccca gttcgggaga cccaagtcaa
      601 ctcctgagac ctgattgtgc acatcgggaa ggtgaacttc aacaaaatcc tcagcctcgt
      661 caccgtcctt gaaaacggta gacgggtctg ccagggggtg cacaaggggg ataaccgagg
      721 attccaacaa ctccatatgt tgctggagga gggatgattg catctctggg attaagacat
      781 tgccgtcagg tcctaatatt ataccattga aaaacacccc gttcacatga ggatgacacc
      841 tccccccaac tcttaaacac ccttttgaag ggaggatctc attccaagtt ctgactgact
      901 tgtagtgagc atcggcttcc atcaaggtct tgttgaatat ggtatatgct tttccaaacc
      961 cagggacaag ttttcttaaa tgactgagac gtctgaaact cactgacttg gttgtcatga
     1021 tggactctag tgcatccaga cactcctctc tcttcctgac caactcctct acaacaaggt
     1081 gctcaatttc gtctgagcga aagtcgtgca ggttcaccaa cttatcggga gggcaccatt
     1141 tggtttcatt tgatgtttgc atcgagaccc atgttccatc cataagtcta agtcctagaa
     1201 ctccacataa cttgagtttg catgctcctt ttaaagactt atataggcct ctttcatcta
     1261 caaagccgca agtctcactc cctttggatg ctctcttccc tctactattg gtaaaaatgt
     1321 cacaagacat ccctagtctc ggattctcgg gcatccaaat ggtgtaatcg tggttagtgg
     1381 agcagtaggt agaagacacc gctactcctg agcacttccc gctagggaag accctcgagt
     1441 gaagggatct gtcatatggg tccaaatctg ccacacttgg agatatgata acgagagact
     1501 ccttggtggt ttttacagtt cgaagccagc ggtagtcagg gtacggattg tgtagagact
     1561 cttcatatct ggggtcaccg gccatcttcc agttgtacgc ggctctacat gcatctggtg
     1621 ttgggcggaa atgctttctt ttgaacgtgg ttgtgacata accaacgaag ttagtgtagg
     1681 tttcagcctc cgtcacaacg cctgtgcaag tgaacccgtt cacttttatg gctaagatgt
     1741 atccaacttt aagttccatg taggagaacc ctgacaggtt ggtgcatcct tcgtcctcca
     1801 ctaccaaatt gtttgggcag ctgaggtgat gtatgtcaat cggactccag ggaccaagct
     1861 tgtctggtat cgtgtaaata gggaatttcc caaaacacaa tggaaaaacc agaaggggta
     1921 caaacaggag agcctgagga accatgggtc cagggttctc ctccacgtct ccagcctgct
     1981 tcagcaggct gaagttagta gctccggatc ccttgtacag ctcgtccatg ccgagagtga
     2041 tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg ttggggtctt
     2101 tgctcagggc ggactgggtg ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc
     2161 cgatgggggt gttctgctgg tagtggtcgg cgagctgcac gctgccgtcc tcgatgttgt
     2221 ggcggatctt gaagttcacc ttgatgccgt tcttctgctt gtcggccatg atatagacgt
     2281 tgtggctgtt gtagttgtac tccagcttgt gccccaggat gttgccgtcc tccttgaagt
     2341 cgatgccctt cagctcgatg cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc
     2401 gggtcttgta gttgccgtcg tccttgaaga agatggtgcg ctcctggacg tagccttcgg
     2461 gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc ggggtagcgg ctgaagcact
     2521 gcacgccgta ggtcagggtg gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg
     2581 tgcagatgaa cttcagggtc agcttgccgt aggtggcatc gccctcgccc tcgccggaca
     2641 cgctgaactt gtggccgttt acgtcgccgt ccagctcgac caggatgggc accaccccgg
     2701 tgaacagctc ctcgcccttg ctcaccatgg gtccagggtt ctcctccacg tctccagcct
     2761 gcttcagcag gctgaagtta gtagctccgg atccggagaa caagtctgcc tggctcttgt
     2821 caggcaccag cagctccttg gatgcgcttt caagactgaa gatgtcagac ctgctttttg
     2881 ctttggactt cccccatgca gcgataccac ccacagctac caggcagcac aggagcactg
     2941 cagtgatcag catccacatg cggccgtgat tcctggctgg cagagcacgt ccaggagcag
     3001 gggcagtggg agcctctgtg ccgttgtccg tgggcaccgc ggggatcgcg ctggtcccgc
     3061 agccccactc gtcccgcccg tcgtcgcagt cggggtgtcc gtcgcacagc cagtcctgcg
     3121 ggtaacactc cccgtgggca ccgggcggct ccgagcagcg gaactgaccg ggggggcaac
     3181 gggacaaaga accgttaccg gacccgttac cggtcacgtt accgggcagc agcagcagca
     3241 gcagcgcggg cagcagccgc gcacctaata acttcgtata gcatacatta tacgaagtta
     3301 tttgccttaa cccagaaatt atcactgtta ttctttagaa tggtgcaaag aataacttcg
     3361 tataggatac tttatacgaa gttatgaatc aacctctgga ttacaaaatt tgtgaaagat
     3421 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     3481 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     3541 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     3601 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     3661 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     3721 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     3781 agctgacgtc ctttccatgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     3841 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     3901 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     3961 gggccgcctc cccgcctgga tcctgtgcct tctagttgcc agccatctgt tgtttgcccc
     4021 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     4081 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     4141 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     4201 tctatggcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4261 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4321 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     4381 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     4441 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4501 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4561 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4621 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4681 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4741 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     4801 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4861 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4921 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4981 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     5041 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     5101 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     5161 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5221 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     5281 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5341 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5401 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5461 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5521 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5581 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5641 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5701 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5761 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5821 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5881 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5941 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     6001 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     6061 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     6121 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     6181 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     6241 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6301 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6361 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6421 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6481 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6541 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6601 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6661 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6721 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6781 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6841 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6901 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg
     6961 cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc
     7021 agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcta
     7081 gactgcagag ggccctgcgt atgagtgcaa gtgggtttta ggaccaggat gaggcggggt
     7141 gggggtgcct acctgacgac cgaccccgac ccactggaca agcacccaac ccccattccc
     7201 caaattgcgc atcccctatc agagaggggg aggggaaaca ggatgcggcg aggcgcgtgc
     7261 gcactgccag cttcagcacc gcggacagtg ccttcgcccc cgcctggcgg cgcgcgccac
     7321 cgccg
//
