LOCUS       sequence-344881-        7326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-on (DIO) helpers for N2c rabies monosynaptic tracing; TVA, eGFP 
            and N2cG under Synpasin promotoer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7326)
  AUTHORS   Pouchelon G, Dwivedi D, Bollmann Y, Agba CK, Xu Q, Mirow AMC, Kim S,
            Qiu Y, Sevier E, Ritola KD, Cossart R, Fishell G
  TITLE     The organization and development of cortical interneuron presynaptic
            circuits are area specific.
  JOURNAL   Cell Rep. 2021 Nov 9;37(6):109993. doi: 
            10.1016/j.celrep.2021.109993.
  PUBMED    34758329
REFERENCE   2  (bases 1 to 7326)
  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: "Cell Rep.";
            date: "2021-11-9"; volume: "37(6):109993. doi"; pages: " 
            10.1016/j.celrep.2021.109993"
COMMENT     Sequence Label: pAAV-VTKS2-TVA-eGFP-N2cG
FEATURES             Location/Qualifiers
     source          1..7326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   36..165
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        193..640
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      647..656
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    662..695
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    746..779
                     /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(2361..2417)
                     /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(2427..3137,3138..3140,3141..3143))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2427..3137,3138..3140,3141..3143))
                     /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(2469..2490)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2818..2837
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3077..3098
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(3144..3200)
                     /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(3683..3716)
                     /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(3767..3800)
                     /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    3802..4390
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3855..3875)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4273..4284)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4398..4622
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4624..4764
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4624..4753
                     /label=AAV2 ITR
     rep_origin      4839..5294
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4926..4945)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5136..5157
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5311..5330)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5430..5452
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5490..5508)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5576..5680
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5681..5749,5750..6541)
                     /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(5899..5918)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6712..7300
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7201..7220
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttcctggcc ttttgctggc cttttgctca catgtcctgc aggcagctgc gcgctcgctc
       61 gctcactgag gccgcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc
      121 gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgctag actgcagagg
      181 gccctgcgta tgagtgcaag tgggttttag gaccaggatg aggcggggtg ggggtgccta
      241 cctgacgacc gaccccgacc cactggacaa gcacccaacc cccattcccc aaattgcgca
      301 tcccctatca gagaggggga ggggaaacag gatgcggcga ggcgcgtgcg cactgccagc
      361 ttcagcaccg cggacagtgc cttcgccccc gcctggcggc gcgcgccacc gccgcctcag
      421 cactgaaggc gcgctgacgt cactcgccgg tcccccgcaa actccccttc ccggccacct
      481 tggtcgcgtc cgcgccgccg ccggcccagc cggaccgcac cacgcgaggc gcgagatagg
      541 ggggcacggg cgcgaccatc tgcgctgcgg cgccggcgac tcagcgctgc ctcagtctgc
      601 ggtgggcagc ggaggagtcg tgtcgtgcct gagagcgcag tcagccgcca ccatggttaa
      661 tataacttcg tataatgtat gctatacgaa gttatgcaga atggtagctg gattgtagct
      721 gctattagca atatgaaacc tcttaataac ttcgtataaa gtatcctata cgaagttatg
      781 gtaccttaca gtctgatctc acctccactc ttatatgatt cccatgaagg tatgactttt
      841 ccgctttggg aagtgactga cacattcccc cctgtccctc caaaactgcg ttgtttcgat
      901 tctggtcgat tggctcttct gcaccatgtc attagggaaa atatcaacac caggccaatc
      961 atggcccctg cagtcatcaa tacatacttt ccccagttcg ggagacccag gtcaacccct
     1021 gagatctgtt tgtacacatc ggggaggtga acttcaacaa aatcctcagc ctcatcacct
     1081 tctttgaaaa ctgtagaagg gtctgccagg gggtgcatca gggggataac tgaagattcc
     1141 aacaactcca tatgttgctg gaggagggat gattgcatct ctgggattag gacatggtcg
     1201 tcaggcccta atattatacc attgaaaaac accccgttca catgaggatg gcaccttcct
     1261 ccaactttca aacacccttt tgaggggatg atctcattcc aggtccggac tgacttgtag
     1321 tgagcatcag cctccatcaa ggttttgttg aatatggtat atgcttttcc aaaccctggg
     1381 acaagttttc tcaggtgact gagacgtctg aaacttactg acttggtggt catgatggac
     1441 tctaatgcgt ccagacattc ctctcttttc ttgactaact cctccacaac gagatgctca
     1501 atctcgtctg agcgaaagtc gtgcaaattc accaactgat ctggagggca ccatttggtc
     1561 tcatctgatg tttgcatcgc gacccatgtt ccatccataa gtctaagtcc aagaactcca
     1621 cataacttga gcctgcatgc tccttttaga gacttataca ggcctctttc atccacaaag
     1681 ccgcaagtct tgttcccgtt ggatgctctc ttccctctgc tattggtaaa aatgtcacaa
     1741 ggtgtccttg gtctcggatt ctcgggcatc caaatggtgt aatcatggtt agttgagcag
     1801 taggtagagg acaccgttat tcctgagcac tttccgccag ggaagaccct tgagtgaagg
     1861 gatttgtcat atgggtccaa atctgtcaca cttggggata tgataatgag ggactctttg
     1921 gtggttctta cagttcgaag ccagtggtag tcggggtatg gattgtgtag ggactcttca
     1981 tatctggggt caccggccat cttccagtta tacgcggctc tacatgcgtc tggggtgggg
     2041 cggaaatgct ttctcttgaa tgtggttgtg acataaccaa caaagttggt gtaggtctct
     2101 gcctctgtca caacacctgt gcaagtgaac ccgttcactt tgatggctga gatgtatccc
     2161 actttgagtt ccatgtagga gaactcggac aggttggtac atccttcatc ctccacaacc
     2221 aggttatttg gacagctgag atggtgtatg tcaatagggc tccagggacc aagttcgtct
     2281 ggtatcgtgt aaatggggaa cttcccgaaa cacaacgaaa aacccagaag gagtacaaac
     2341 aaaagaacct gaggaaccat aggtccaggg ttctcctcca cgtctccagc ctgcttcagc
     2401 aggctgaagt tagtagctcc gcttcccttg tacagctcgt ccatgccgag agtgatcccg
     2461 gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc
     2521 agggcggact gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg
     2581 ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg
     2641 atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg
     2701 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
     2761 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
     2821 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg
     2881 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
     2941 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
     3001 atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg
     3061 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
     3121 agctcctcgc ccttgctcac catgggtcca gggttctcct ccacgtctcc agcctgcttc
     3181 agcaggctga agttagtagc tccggatccg gagaacaagt ctgcctggct cttgtcaggc
     3241 accagcagct ccttggatgc gctttcaaga ctgaagatgt cagacctgct ttttgctttg
     3301 gacttccccc atgcagcgat accacccaca gctaccaggc agcacaggag cactgcagtg
     3361 atcagcatcc acatgcggcc gtgattcctg gctggcagag cacgtccagg agcaggggca
     3421 gtgggagcct ctgtgccgtt gtccgtgggc accgcgggga tcgcgctggt cccgcagccc
     3481 cactcgtccc gcccgtcgtc gcagtcgggg tgtccgtcgc acagccagtc ctgcgggtaa
     3541 cactccccgt gggcaccggg cggctccgag cagcggaact gaccgggggg gcaacgggac
     3601 aaagaaccgt taccggaccc gttaccggtc acgttaccgg gcagcagcag cagcagcagc
     3661 gcgggcagca gccgcgcacc taataacttc gtatagcata cattatacga agttatttgc
     3721 cttaacccag aaattatcac tgttattctt tagaatggtg caaagaataa cttcgtatag
     3781 gatactttat acgaagttat gaatcaacct ctggattaca aaatttgtga aagattgact
     3841 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3901 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     3961 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     4021 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     4081 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     4141 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg
     4201 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     4261 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     4321 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     4381 gcctccccgc ctggatcctg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     4441 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     4501 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     4561 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     4621 ggcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     4681 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     4741 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     4801 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     4861 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     4921 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4981 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     5041 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     5101 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     5161 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     5221 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     5281 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5341 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5401 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5461 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     5521 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     5581 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5641 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5701 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5761 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5821 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5881 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5941 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6001 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6061 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6121 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6181 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6241 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6301 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6361 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6421 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6481 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6541 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6601 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6661 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6721 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6781 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6841 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6901 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6961 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     7021 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     7081 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     7141 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     7201 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     7261 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     7321 tttacg
//
