LOCUS       sequence-469834-        8065 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pAAV transfer plasmid with CAG promoter driving Flp-dependent 
            expression of mKate2.0 and N2c G protein separated by a T2A linker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8065)
  AUTHORS   Mendelsohn AI, Nikoobakht L, Bikoff JB, Costa RM
  TITLE     Segregated basal ganglia output pathways correspond to genetically 
            divergent neuronal subclasses.
  JOURNAL   bioRxiv [Preprint]. 2024 Sep 18:2024.08.28.610136. doi: 
            10.1101/2024.08.28.610136.
  PUBMED    39257765
REFERENCE   2  (bases 1 to 8065)
  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: "bioRxiv 
            [Preprint]. 2024 Sep 18:2024.08.28.610136. doi: 
            10.1101/2024.08.28.610136."
COMMENT     Sequence Label: pAAV-CAG-FlpX-mKate2.0-N2cG
FEATURES             Location/Qualifiers
     source          1..8065
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(830..850)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        835..1164
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1015..1150
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1374..1598
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1587..1604
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     misc_feature    1631..2219
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1737..1748
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     2146..2166
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    2258..2291
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     protein_bind    2379..2412
                     /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)"
     regulatory      2455..2464
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2461..3156
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             3172..3225
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    complement(4820..4853)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     protein_bind    complement(4941..4974)
                     /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)"
     primer_bind     complement(5009..5028)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(5090..5112)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(5391..5668)
                     /label=chicken beta-actin promoter
     repeat_region   5974..6103
                     /label=AAV2 ITR
     promoter        complement(6124..6142)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(6125..6142)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(6432..6451)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(6665..6684)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6784..6806
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6844..6862)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6930..7034
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7035..7103,7104..7895)
                     /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(7253..7272)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgtg gaatgtgtgt
      841 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
      901 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
      961 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     1021 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     1081 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     1141 tttggaggcc taggcttttg caaaaagctc tgcgcgctcg ctcgctcact gaggccgccc
     1201 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
     1261 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
     1321 tctacgtagc catgctctag tcgagcccca gctggttctt tccgcctcag aagccataga
     1381 gcccaccgca tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg
     1441 ccccacccca ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc
     1501 attttattag gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga
     1561 ggggcaaaca acagatggct ggcaactaga aggcacagtc gaggctgatc agcgagtcga
     1621 cggtatcgat gcggggaggc ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga
     1681 agacgcggaa gaggccgcag agccggcagc aggccgcggg aaggaaggtc cgctggattg
     1741 agggccgaag ggacgtagca gaaggacgtc ccgcgcagaa tccaggtggc aacacaggcg
     1801 agcagccaag gaaaggacga tgatttcccc gacaacacca cggaattgtc agtgcccaac
     1861 agccgagccc ctgtccagca gcgggcaagg caggcggcga tgagttccgc cgtggcaata
     1921 gggaggggga aagcgaaagt cccggaaagg agctgacagg tggtggcaat gccccaacca
     1981 gtgggggttg cgtcagcaaa cacagtgcac accacgccac gttgcctgac aacgggccac
     2041 aactcctcat aaagagacag caaccaggat ttatacaagg aggagaaaat gaaagccata
     2101 cgggaagcaa tagcatgata caaaggcatt aaagcagcgt atccacatag cgtaaaagga
     2161 gcaacatagt taagaatacc agtcaatctt tcacaaattt tgtaatccag aggttgatta
     2221 tcgataagct gttaaagctt gaacctcttc gagggacgaa gttcctattc ttcaaatagt
     2281 ataggaactt cacccggatt caaagttagg gtcgatggtg agatctggac tagagggtcg
     2341 atggtgatgc ttggatcaaa ataggaagaa tcccaatgga agttcctatt ctctagaaag
     2401 tataggaact tcgatatcag atctctcgag gcggccgcac tagtgagctc gtttgccacc
     2461 atggtgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg
     2521 aacaaccacc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag
     2581 accatgagaa tcaaggcggt cgagggcggc cctctcccct tcgccttcga catcctggct
     2641 accagcttca tgtacggcag caaaaccttc atcaaccaca cccagggcat ccccgacttc
     2701 tttaagcagt ccttccccga gggcttcaca tgggagagag tcaccacata cgaagacggg
     2761 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc
     2821 aagatcagag gggtgaactt cccatccaac ggccctgtga tgcagaagaa aacactcggc
     2881 tgggaggcct ccaccgagac cctgtacccc gctgacggcg gcctggaagg cagagccgac
     2941 atggccctga agctcgtggg cgggggccac ctgatctgca acttgaagac cacatacaga
     3001 tccaagaaac ccgctaagaa cctcaagatg cccggcgtct actatgtgga cagaagactg
     3061 gaaagaatca aggaggccga caaagagacc tacgtcgagc agcacgaggt ggctgtggcc
     3121 agatactgcg acctccctag caaactgggg cacagagcta gcggtagtgg agagggcaga
     3181 ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaacgcg tgttcctcag
     3241 gttcttttgt ttgtactcct tctgggtttt tcgttgtgtt tcgggaagtt ccccatttac
     3301 acgataccag acgaacttgg tccctggagc cctattgaca tacaccatct cagctgtcca
     3361 aataacctgg ttgtggagga tgaaggatgt accaacctgt ccgagttctc ctacatggaa
     3421 ctcaaagtgg gatacatctc agccatcaaa gtgaacgggt tcacttgcac aggtgttgtg
     3481 acagaggcag agacctacac caactttgtt ggttatgtca caaccacatt caagagaaag
     3541 catttccgcc ccaccccaga cgcatgtaga gccgcgtata actggaagat ggccggtgac
     3601 cccagatatg aagagtccct acacaatcca taccccgact accactggct tcgaactgta
     3661 agaaccacca aagagtccct cattatcata tccccaagtg tgacagattt ggacccatat
     3721 gacaaatccc ttcactcaag ggtcttccct ggcggaaagt gctcaggaat aacggtgtcc
     3781 tctacctact gctcaactaa ccatgattac accatttgga tgcccgagaa tccgagacca
     3841 aggacacctt gtgacatttt taccaatagc agagggaaga gagcatccaa cgggaacaag
     3901 acttgcggct ttgtggatga aagaggcctg tataagtctc taaaaggagc atgcaggctc
     3961 aagttatgtg gagttcttgg acttagactt atggatggaa catgggtcgc gatgcaaaca
     4021 tcagatgaga ccaaatggtg ccctccagat cagttggtga atttgcacga ctttcgctca
     4081 gacgagattg agcatctcgt tgtggaggag ttagtcaaga aaagagagga atgtctggac
     4141 gcattagagt ccatcatgac caccaagtca gtaagtttca gacgtctcag tcacctgaga
     4201 aaacttgtcc cagggtttgg aaaagcatat accatattca acaaaacctt gatggaggct
     4261 gatgctcact acaagtcagt ccggacctgg aatgagatca tcccctcaaa agggtgtttg
     4321 aaagttggag gaaggtgcca tcctcatgtg aacggggtgt ttttcaatgg tataatatta
     4381 gggcctgacg accatgtcct aatcccagag atgcaatcat ccctcctcca gcaacatatg
     4441 gagttgttgg aatcttcagt tatccccctg atgcaccccc tggcagaccc ttctacagtt
     4501 ttcaaagaag gtgatgaggc tgaggatttt gttgaagttc acctccccga tgtgtacaaa
     4561 cagatctcag gggttgacct gggtctcccg aactggggaa agtatgtatt gatgactgca
     4621 ggggccatga ttggcctggt gttgatattt tccctaatga catggtgcag aagagccaat
     4681 cgaccagaat cgaaacaacg cagttttgga gggacagggg ggaatgtgtc agtcacttcc
     4741 caaagcggaa aagtcatacc ttcatgggaa tcatataaga gtggaggtga gatcagactg
     4801 taaaaacctg caggaattcg aagttcctat actatttgaa gaataggaac ttcacgggtc
     4861 aatctgaaac ggtcaagagg gtcgatggtg atgcttggca attcgggtcg atggtgaagc
     4921 attggtcttc ctattttgat gaagttccta tactttctag agaataggaa cttcggggat
     4981 ccaattcttt gccaaaatga tgagacagca caataaccag cacgttgccc aggagctgta
     5041 ggaaaaagaa gaaggcatga acatggttag cagaggctct agagccgccg gtcacacgcc
     5101 agaagccgaa ccccgccctg ccccgtcccc cccgaaggca gccgtccccc cgcggacagc
     5161 cccgagccgc tccccccgca caaagggccc tcccggagcc cctcaaggct ttcacgcagc
     5221 cacagaaaag aaacaagccg tcattaaacc aagcgctaat tacagcccgg aggagaaggg
     5281 ccgtcccgcc cgctcacctg tgggagtaac gcggtcagtc agagccgggg cgggcggcgc
     5341 gaggcggcgg cggagcgggg cacggggcga aggcagcgcg cagcgactcc cgcccgccgc
     5401 gcgcttcgct ttttataggg ccgccgccgc cgccgcctcg ccataaaagg aaactttcgg
     5461 agcgcgccgc tctgattggc tgccgccgca cctctccscc tcsccccscc ccccccctcc
     5521 cccccccccc ccccccctgg cgcgcgcccc cccccccccc ccgcccccat cgctgcacaa
     5581 aataattaaa aaataaataa atacaaaatt gggggtgggg agggggggga gatggggaga
     5641 gtgaagcaga acgtggggct cacctcgacc atggtaatag cgatgactaa tacgtagatg
     5701 tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat
     5761 ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc
     5821 aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc
     5881 gttactatgg gaacatacgt cattattgac gtcaatggta cctagagcat ggctacgtag
     5941 ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca
     6001 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     6061 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctagaat taattccgtg
     6121 tattctatag tgtcacctaa atcgtatgtg tatgatacat aaggttatgt attaattgta
     6181 gccgcgttct aacgacaata tgtacaagcc taattgtgta gcatctggct tactgaagca
     6241 gaccctatca tctctctcgt aaactgccgt cagagtcggt ttggttggac gaaccttctg
     6301 agtttctggt aacgccgtcc cgcacccgga aatggtcagc gaaccaatca gcagggtcat
     6361 cgctagccag atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc
     6421 ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg
     6481 gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt
     6541 gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct
     6601 actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgaa tggtgcactc
     6661 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     6721 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     6781 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     6841 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     6901 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     6961 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     7021 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     7081 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     7141 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     7201 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     7261 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     7321 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     7381 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     7441 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     7501 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     7561 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     7621 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     7681 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     7741 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     7801 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     7861 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     7921 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     7981 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     8041 ccgtagaaaa gatcaaagga tcttc
//
