LOCUS       sequence-193696-        6521 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  helper virus for monosynaptic tracing; to be coinjected with 
            pAAV-TREtight-mTagBFP2-B19G.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6521)
  AUTHORS   Liu K, Kim J, Kim DW, Zhang YS, Bao H, Denaxa M, Lim SA, Kim E, Liu 
            C, Wickersham IR, Pachinis V, Hattar S, Song J, Brown SP, Blackshaw 
            S
  TITLE     Lhx6-positive GABA-releasing neurons of the zona incerta promote 
            sleep.
  JOURNAL   Nature. 2017 Aug 31;548(7669):582-587. doi: 10.1038/nature23663. 
            Epub 2017 Aug 23.
  PUBMED    28847002
REFERENCE   2  (bases 1 to 6521)
  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/nature23663"; journalName: "Nature"; date: "2017-08-31- 
            31"; volume: "548"; issue: "7669"; pages: "582-587"
COMMENT     Sequence Label: pAAV-syn-FLEX-splitTVA-EGFP-tTA
FEATURES             Location/Qualifiers
     source          1..6521
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        158..605
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      612..621
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    627..660
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    711..744
                     /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(900..1520)
                     /codon_start=1
                     /gene="synthetic gene"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     CDS             complement(1521..1577)
                     /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(1587..2297,2298..2300,2301..2303))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1587..2297,2298..2300,2301..2303))
                     /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(1629..1650)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1978..1997
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2237..2258
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2304..2360)
                     /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(2843..2876)
                     /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(2927..2960)
                     /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    2962..3550
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3015..3035)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3433..3444)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3558..3782
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3784..3924
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3784..3913
                     /label=AAV2 ITR
     rep_origin      3999..4454
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4086..4105)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4296..4317
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4471..4490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4590..4612
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4650..4668)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4736..4840
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4841..4909,4910..5701)
                     /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(5059..5078)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5872..6460
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6361..6380
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gctagactgc agagggccct gcgtatgagt gcaagtgggt tttaggacca
      181 ggatgaggcg gggtgggggt gcctacctga cgaccgaccc cgacccactg gacaagcacc
      241 caacccccat tccccaaatt gcgcatcccc tatcagagag ggggagggga aacaggatgc
      301 ggcgaggcgc gtgcgcactg ccagcttcag caccgcggac agtgccttcg cccccgcctg
      361 gcggcgcgcg ccaccgccgc ctcagcactg aaggcgcgct gacgtcactc gccggtcccc
      421 cgcaaactcc ccttcccggc caccttggtc gcgtccgcgc cgccgccggc ccagccggac
      481 cgcaccacgc gaggcgcgag ataggggggc acgggcgcga ccatctgcgc tgcggcgccg
      541 gcgactcagc gctgcctcag tctgcggtgg gcagcggagg agtcgtgtcg tgcctgagag
      601 cgcagtcagc cgccaccatg gttaatataa cttcgtataa tgtatgctat acgaagttat
      661 gcagaatggt agctggattg tagctgctat tagcaatatg aaacctctta ataacttcgt
      721 ataaagtatc ctatacgaag ttatggcgcg cctcacagca tatccaggtc aaaatcatca
      781 agtgcatcag cagggagcat gtccaaatca aagtcatcca gggcatcagc aggcagcatg
      841 tccaggtcaa aatcatccaa ggcatcagca ggcacgtgct ttctcttttt gggcacgtga
      901 gagccagact cacatttcag ctgcttttcc aggccacaga tgatcagttc caggccaaaa
      961 aggaatgcag gctcagcccc ttggtggtca aacaactcaa tagcttgccg cagcagtggt
     1021 ggcatggagt cagtggtggg ggtctccctc tcctccttgg caacttggtg ctcctggtcc
     1081 tccagcacac agcccaaggt gaagtggccc acagcagaga gggcatacaa ggcattttcc
     1141 agggagaagc cttgttggca caggaatgcc agctggttct ccagggtctc atactgcttc
     1201 tctgtaggcc tggtgcccaa gtgaactttg gcaccatccc ggtgggagag caaggcacat
     1261 ctgaagctct tggcattgtt cctcaggaag tcctgccagg actccccttc cagagggcag
     1321 aagtgggtgt ggtgcctgtc caacatctca atggccaggg catcaagcag agccctcttg
     1381 ttcttcacat gccagtacag ggtaggctgc tccacaccca gcttctgggc cagcttcctg
     1441 gtggtcaagc cctcaatgcc aacttcattc aggagctcca aggcagagtt gatgactttg
     1501 ctcttgtcca gtctggacat aggtccaggg ttctcctcca cgtctccagc ctgcttcagc
     1561 aggctgaagt tagtagctcc gcttcccttg tacagctcgt ccatgccgag agtgatcccg
     1621 gcggcggtca cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc
     1681 agggcggact gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg
     1741 ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg
     1801 atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg
     1861 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
     1921 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
     1981 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg
     2041 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
     2101 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
     2161 atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg
     2221 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
     2281 agctcctcgc ccttgctcac cataggtcca gggttctcct ccacgtctcc agcctgcttc
     2341 agcaggctga agttagtagc tccgcttccg gagaacaagt ctgcctggct cttgtcaggc
     2401 accagcagct ccttggatgc gctttcaaga ctgaagatgt cagacctgct ttttgctttg
     2461 gacttccccc atgcagcgat accacccaca gctaccaggc agcacaggag cactgcagtg
     2521 atcagcatcc acatgcggcc gtgattcctg gctggcagag cacgtccagg agcaggggca
     2581 gtgggagcct ctgtgccgtt gtccgtgggc accgcgggga tcgcgctggt cccgcagccc
     2641 cactcgtccc gcccgtcgtc gcagtcgggg tgtccgtcgc acagccagtc ctgcgggtaa
     2701 cactccccgt gggcaccggg cggctccgag cagcggaact gaccgggggg gcaacgggac
     2761 aaagaaccgt taccggaccc gttaccggtc acgttaccgg gcagcagcag cagcagcagc
     2821 gcgggcagca gccgcgcacc taataacttc gtatagcata cattatacga agttatttgc
     2881 cttaacccag aaattatcac tgttattctt tagaatggtg caaagaataa cttcgtatag
     2941 gatactttat acgaagttat gaatcaacct ctggattaca aaatttgtga aagattgact
     3001 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3061 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     3121 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     3181 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     3241 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     3301 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg
     3361 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     3421 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     3481 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     3541 gcctccccgc ctggatcctg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3601 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3661 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3721 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3781 ggcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     3841 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     3901 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     3961 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     4021 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     4081 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     4141 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4201 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4261 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4321 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     4381 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4441 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4501 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4561 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4621 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     4681 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     4741 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4801 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4861 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4921 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4981 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5041 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5101 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5161 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5221 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5281 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5341 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5401 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5461 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5521 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     5581 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5641 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     5701 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     5761 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5821 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5881 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5941 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6001 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6061 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6121 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6181 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6241 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6301 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6361 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6421 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6481 tttacggttc ctggcctttt gctggccttt tgctcacatg t
//
