LOCUS       sequence-222091-        6218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG-FLEx-iBARK-p2A-mCherry: Viral expression vector for 
            Cre-dependent Gaq silencing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6218)
  AUTHORS   Nagai J, Bellafard A, Qu Z, Yu X, Ollivier M, Gangwani MR, 
            Diaz-Castro B, Coppola G, Schumacher SM, Golshani P, Gradinaru V, 
            Khakh BS
  TITLE     Specific and behaviorally consequential astrocyte Gq GPCR signaling 
            attenuation in vivo with ibetaARK.
  JOURNAL   Neuron. 2021 Jul 21;109(14):2256-2274.e9. doi: 
            10.1016/j.neuron.2021.05.023. Epub 2021 Jun 16.
  PUBMED    34139149
REFERENCE   2  (bases 1 to 6218)
  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: 
            "10.1016/j.neuron.2021.05.023"; journalName: "Neuron"; date: 
            "2021-07-21- 21"; volume: "109"; issue: "14"; pages: "2256-2274.e9"
COMMENT     Sequence Label: CAG FLEX BARK p2A mCherry
FEATURES             Location/Qualifiers
     source          1..6218
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   56..185
                     /label=AAV2 ITR
     polyA_signal    347..468
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(384..403)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     438..457
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    511..1099
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             617..628
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1026..1046
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(1124..1157)
                     /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(1208..1241)
                     /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             1272..1298
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1299..1322
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1755..1811
                     /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             1812..2522
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1957..1975)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2225..2244
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    2531..2564
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2615..2648
                     /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)."
     primer_bind     complement(2705..2724)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(2741..2763)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(3043..3320)
                     /label=chicken beta-actin promoter
     repeat_region   3631..3760
                     /label=AAV2 ITR
     rep_origin      3832..4287
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3919..3938)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4129..4150
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4313..4417
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4418..4486,4487..5278)
                     /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(4636..4655)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5449..6037
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5938..5957
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg
       61 cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg
      121 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg
      181 ttccttgtag ttaatgatta acccgccatg ctacttatct acgtagccat gctctaggaa
      241 gatccttatc gattttacca catttgtaga ggttttactt gctttaaaaa acctcccaca
      301 tctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
      361 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
      421 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgctc
      481 gaagcggccg cccgggtcga cggtatcgat gcggggaggc ggcccaaagg gagatccgac
      541 tcgtctgagg gcgaaggcga agacgcggaa gaggccgcag agccggcagc aggccgcggg
      601 aaggaaggtc cgctggattg agggccgaag ggacgtagca gaaggacgtc ccgcgcagaa
      661 tccaggtggc aacacaggcg agcagccaag gaaaggacga tgatttcccc gacaacacca
      721 cggaattgtc agtgcccaac agccgagccc ctgtccagca gcgggcaagg caggcggcga
      781 tgagttccgc cgtggcaata gggaggggga aagcgaaagt cccggaaagg agctgacagg
      841 tggtggcaat gccccaacca gtgggggttg cgtcagcaaa cacagtgcac accacgccac
      901 gttgcctgac aacgggccac aactcctcat aaagagacag caaccaggat ttatacaagg
      961 aggagaaaat gaaagccata cgggaagcaa tagcatgata caaaggcatt aaagcagcgt
     1021 atccacatag cgtaaaagga gcaacatagt taagaatacc agtcaatctt tcacaaattt
     1081 tgtaatccag aggttgatta tcgataagct tgatatcgaa ttcataactt cgtatagcat
     1141 acattatacg aagttattct ttgcaccatt ctaaagaata acagtgataa tttctgggtt
     1201 aaggcaaata acttcgtata ggatacttta tacgaagtta tgctagcggg agacccaagc
     1261 ttaccaccat gtacccatac gatgttcctg actatgcgga ctacaaagac gatgacgaca
     1321 agaagtacct ggaggaccgg ggcgaggtga cttttgagaa gatcttctcc cagaagctgg
     1381 ggtacctgct tttccgagac ttctgcctga agcacctgga ggaggccaag cccttggtag
     1441 agttctacga ggagatcaag aaatacgaga agctggagac agaggaggag cgcctggtct
     1501 gcagccgaga gatcttcgac acgtacatca tgaaggagct gctggcctgc tcacatcctt
     1561 tctcgaagag cgccattgag cacgtccagg gccatctggt gaagaagcag gtgcctccgg
     1621 atctcttcca gccatatatt gaagaaattt gccagaacct ccgaggagac gtgttccaga
     1681 aattcatcga gagcgataaa ttcacacgat tttgccagtg gaagaatgta gagctcaaca
     1741 tccacggtag tggcgcaaca aacttctctc tgctgaaaca agccggagat gtcgaagaga
     1801 atcctggacc gatggtgagc aagggcgagg aggataacat ggccatcatc aaggagttca
     1861 tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     1921 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     1981 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct
     2041 acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca
     2101 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct
     2161 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     2221 acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc
     2281 ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
     2341 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg
     2401 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     2461 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     2521 agggcgcgcc ataacttcgt ataatgtatg ctatacgaag ttattaagag gtttcatatt
     2581 gctaatagca gctacaatcc agctaccatt ctgcataact tcgtataaag tatcctatac
     2641 gaagttattc cggagtcgac tctagaggat cccgatccaa ttctttgcca aaatgatgag
     2701 acagcacaat aaccagcacg ttgcccagga gagagccgcc ggtcacacgc cagaagccga
     2761 accccgccct gccccgtccc ccccgaaggc agccgtcccc ccgcggacag ccccgagccg
     2821 ctccccccgc acaaagggcc ctcccggagc ccctcaaggc tttcacgcag ccacagaaaa
     2881 gaaacaagcc gtcattaaac caagcgctaa ttacagcccg gaggagaagg gccgtcccgc
     2941 ccgctcacct gtgggagtaa cgcggtcagt cagagccggg gcgggcggcg cgaggcggcg
     3001 gcggagcggg gcacggggcg aaggcagcgt cgcagcgact cccgcccgcc gcgcgcttcg
     3061 ctttttatag ggccgccgcc gccgccgcct cgccataaaa ggaaactttc ggagcgcgcc
     3121 gctctgattg gctgccgccg cacctctccg cctcgccccg ccccgcccct cgccccgccc
     3181 cgccccgcct ggcgcgcgcc cccccccccc ccccgccccc atcgctgcac aaaataatta
     3241 aaaaataaat aaatacaaaa ttgggggtgg ggaggggggg gagatgggga gagtgaagca
     3301 gaacgtgggg ctcacctcga ccatggtaat agcgatgact aatacgtaga tgtactgcca
     3361 agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc
     3421 attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc
     3481 agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg gcgttactat
     3541 gggaacatac gtcattattg acgtcaatgg tactcttcct agagcatggc tacgtagata
     3601 agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc
     3661 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     3721 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctggcgtaat agcgaagagg
     3781 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct
     3841 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3901 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3961 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     4021 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     4081 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     4141 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     4201 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     4261 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     4321 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4381 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     4441 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     4501 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     4561 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     4621 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4681 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4741 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4801 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4861 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4921 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     4981 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     5041 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     5101 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     5161 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     5221 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     5281 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     5341 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     5401 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     5461 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     5521 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     5581 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     5641 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5701 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5761 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5821 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5881 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5941 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     6001 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     6061 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     6121 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     6181 gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
