LOCUS       sequence-222092-        6203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG-FLEx-iBARK(D110A)-p2A-mCherry: Negative control viral expression
            vector for Cre-dependent Gaq silencing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6203)
  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 6203)
  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 D110A p2A mCherry
FEATURES             Location/Qualifiers
     source          1..6203
                     /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             1257..1283
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1284..1307
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1740..1796
                     /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             1797..2507
                     /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(1942..1960)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2210..2229
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    2516..2549
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2600..2633
                     /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(2690..2709)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(2726..2748)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(3028..3305)
                     /label=chicken beta-actin promoter
     repeat_region   3616..3745
                     /label=AAV2 ITR
     rep_origin      3817..4272
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3904..3923)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4114..4135
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4298..4402
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4403..4471,4472..5263)
                     /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(4621..4640)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5434..6022
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5923..5942
                     /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 tgctagcacc accatgtacc
     1261 catacgatgt tcctgactat gcggactaca aagacgatga cgacaagaag tacctggagg
     1321 accggggcga ggtgactttt gagaagatct tctcccagaa gctggggtac ctgcttttcc
     1381 gagacttctg cctgaagcac ctggaggagg ccaagccctt ggtagagttc tacgaggaga
     1441 tcaagaaata cgagaagctg gagacagagg aggagcgcct ggtctgcagc cgagagatct
     1501 tcgccacgta catcatgaag gagctgctgg cctgctcaca tcctttctcg aagagcgcca
     1561 ttgagcacgt ccagggccat ctggtgaaga agcaggtgcc tccggatctc ttccagccat
     1621 atattgaaga aatttgccag aacctccgag gagacgtgtt ccagaaattc atcgagagcg
     1681 ataaattcac acgattttgc cagtggaaga atgtagagct caacatccac ggtagtggcg
     1741 caacaaactt ctctctgctg aaacaagccg gagatgtcga agagaatcct ggaccgatgg
     1801 tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
     1861 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     1921 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg
     1981 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
     2041 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     2101 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     2161 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     2221 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     2281 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg
     2341 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     2401 tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg
     2461 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtagggc gcgccataac
     2521 ttcgtataat gtatgctata cgaagttatt aagaggtttc atattgctaa tagcagctac
     2581 aatccagcta ccattctgca taacttcgta taaagtatcc tatacgaagt tattccggag
     2641 tcgactctag aggatcccga tccaattctt tgccaaaatg atgagacagc acaataacca
     2701 gcacgttgcc caggagagag ccgccggtca cacgccagaa gccgaacccc gccctgcccc
     2761 gtcccccccg aaggcagccg tccccccgcg gacagccccg agccgctccc cccgcacaaa
     2821 gggccctccc ggagcccctc aaggctttca cgcagccaca gaaaagaaac aagccgtcat
     2881 taaaccaagc gctaattaca gcccggagga gaagggccgt cccgcccgct cacctgtggg
     2941 agtaacgcgg tcagtcagag ccggggcggg cggcgcgagg cggcggcgga gcggggcacg
     3001 gggcgaaggc agcgtcgcag cgactcccgc ccgccgcgcg cttcgctttt tatagggccg
     3061 ccgccgccgc cgcctcgcca taaaaggaaa ctttcggagc gcgccgctct gattggctgc
     3121 cgccgcacct ctccgcctcg ccccgccccg cccctcgccc cgccccgccc cgcctggcgc
     3181 gcgccccccc cccccccccg cccccatcgc tgcacaaaat aattaaaaaa taaataaata
     3241 caaaattggg ggtggggagg ggggggagat ggggagagtg aagcagaacg tggggctcac
     3301 ctcgaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca
     3361 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     3421 gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     3481 ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat
     3541 tattgacgtc aatggtactc ttcctagagc atggctacgt agataagtag catggcgggt
     3601 taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     3661 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     3721 cctcagtgag cgagcgagcg cgcagctggc gtaatagcga agaggcccgc accgatcgcc
     3781 cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa
     3841 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     3901 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     3961 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4021 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4081 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4141 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     4201 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4261 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     4321 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4381 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4441 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     4501 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     4561 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     4621 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     4681 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4741 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     4801 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     4861 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     4921 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     4981 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     5041 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5101 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5161 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5221 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     5281 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5341 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5401 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5461 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5521 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5581 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5641 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5701 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5761 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5821 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5881 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5941 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6001 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6061 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     6121 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     6181 gtcagtgagc gaggaagcgg aag
//
