LOCUS       sequence-193698-        6379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  helper virus for monosynaptic tracing; to be coinjected with 
            pAAV-syn-FLEX-splitTVA-EGFP-tTA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6379)
  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 6379)
  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-TREtight-mTagBFP2-B19G
FEATURES             Location/Qualifiers
     source          1..6379
                     /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        140..454
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    148..166
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    184..202
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    219..237
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    255..273
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    291..309
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    326..344
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    362..380
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     427..451
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             474..1172
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             1182..1238
                     /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"
     misc_feature    2820..3408
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2873..2893)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3291..3302)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3416..3640
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3642..3782
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3642..3771
                     /label=AAV2 ITR
     rep_origin      3857..4312
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3944..3963)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4154..4175
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4329..4348)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4448..4470
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4508..4526)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4594..4698
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4699..4767,4768..5559)
                     /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(4917..4936)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5730..6318
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6219..6238
                     /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 acgcgtctcg agtttactcc ctatcagtga tagagaacgt atgtcgagtt
      181 tactccctat cagtgataga gaacgatgtc gagtttactc cctatcagtg atagagaacg
      241 tatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac tccctatcag
      301 tgatagagaa cgtatgtcga gtttatccct atcagtgata gagaacgtat gtcgagttta
      361 ctccctatca gtgatagaga acgtatgtcg aggtaggcgt gtacggtggg aggcctatat
      421 aagcagagct cgtttagtga accgtcagat cgcctggaga attcgccgcc accatgagcg
      481 agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc
      541 acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa
      601 tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc
      661 tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt
      721 ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga
      781 ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag
      841 gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct
      901 tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga
      961 agctcgtggg cgggagccat ctgatcgcaa acgccaagac cacatataga tccaagaaac
     1021 ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca
     1081 aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg
     1141 acctccctag caaactgggg cacaagctta atggaagcgg agctactaac ttcagcctgc
     1201 tgaagcaggc tggagacgtg gaggagaacc ctggacctat ggttcctcag gctctcctgt
     1261 ttgtacccct tctggttttt ccattgtgtt ttgggaaatt ccctatttac acgataccag
     1321 acaagcttgg tccctggagt ccgattgaca tacatcacct cagctgccca aacaatttgg
     1381 tagtggagga cgaaggatgc accaacctgt cagggttctc ctacatggaa cttaaagttg
     1441 gatacatctt agccataaaa gtgaacgggt tcacttgcac aggcgttgtg acggaggctg
     1501 aaacctacac taacttcgtt ggttatgtca caaccacgtt caaaagaaag catttccgcc
     1561 caacaccaga tgcatgtaga gccgcgtaca actggaagat ggccggtgac cccagatatg
     1621 aagagtctct acacaatccg taccctgact accgctggct tcgaactgta aaaaccacca
     1681 aggagtctct cgttatcata tctccaagtg tggcagattt ggacccatat gacagatccc
     1741 ttcactcgag ggtcttccct agcgggaagt gctcaggagt agcggtgtct tctacctact
     1801 gctccactaa ccacgattac accatttgga tgcccgagaa tccgagacta gggatgtctt
     1861 gtgacatttt taccaatagt agagggaaga gagcatccaa agggagtgag acttgcggct
     1921 ttgtagatga aagaggccta tataagtctt taaaaggagc atgcaaactc aagttatgtg
     1981 gagttctagg acttagactt atggatggaa catgggtctc gatgcaaaca tcaaatgaaa
     2041 ccaaatggtg ccctcccgat aagttggtga acctgcacga ctttcgctca gacgaaattg
     2101 agcaccttgt tgtagaggag ttggtcagga agagagagga gtgtctggat gcactagagt
     2161 ccatcatgac aaccaagtca gtgagtttca gacgtctcag tcatttaaga aaacttgtcc
     2221 ctgggtttgg aaaagcatat accatattca acaagacctt gatggaagcc gatgctcact
     2281 acaagtcagt cagaacttgg aatgagatcc tcccttcaaa agggtgttta agagttgggg
     2341 ggaggtgtca tcctcatgtg aacggggtgt ttttcaatgg tataatatta ggacctgacg
     2401 gcaatgtctt aatcccagag atgcaatcat ccctcctcca gcaacatatg gagttgttgg
     2461 aatcctcggt tatccccctt gtgcaccccc tggcagaccc gtctaccgtt ttcaaggacg
     2521 gtgacgaggc tgaggatttt gttgaagttc accttcccga tgtgcacaat caggtctcag
     2581 gagttgactt gggtctcccg aactggggga agtatgtatt actgagtgca ggggccctga
     2641 ctgccttgat gttgataatt ttcctgatga catgttgtag aagagtcaat cgatcagaac
     2701 ctacgcaaca caatctcaga gggacaggga gggaggtgtc agtcactccc caaagcggga
     2761 agatcatatc ttcatgggaa tcacacaaga gtgggggtga gaccagactg taagctagca
     2821 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     2881 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     2941 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     3001 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     3061 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     3121 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     3181 tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg
     3241 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     3301 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     3361 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct ggatcctgtg
     3421 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     3481 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     3541 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     3601 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg caggaacccc tagtgatgga
     3661 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     3721 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     3781 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     3841 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3901 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     3961 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4021 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4081 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4141 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     4201 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     4261 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     4321 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     4381 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4441 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     4501 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     4561 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     4621 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     4681 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     4741 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     4801 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     4861 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     4921 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     4981 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5041 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5101 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5161 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5221 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5281 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5341 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5401 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5461 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     5521 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     5581 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     5641 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5701 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5761 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5821 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     5881 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5941 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6001 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6061 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6121 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     6181 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6241 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6301 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6361 tggccttttg ctcacatgt
//
