LOCUS       sequence-227783-        6358 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GfaABC1D-iBARK(D110A)-p2A-mCherry: Negative control viral expression
            vector for astrocyte Gaq silencing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6358)
  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 6358)
  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: GfaABC1D BARK D110A p2A mCherry
FEATURES             Location/Qualifiers
     source          1..6358
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   259..388
                     /label=AAV2 ITR
     intron          1239..1371
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1354..1373
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1416..1434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1471..1497
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1498..1521
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1954..2010
                     /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             2011..2721
                     /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(2156..2174)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2424..2443
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2746..3334
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2799..2819)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3217..3228)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3383..3504
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3394..3413)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3448..3467
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3666..3795
                     /label=AAV2 ITR
     primer_bind     complement(3814..3831)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3814..3830)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3823..3845)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3972..4427
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4059..4078)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4269..4290
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4453..4557
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4558..4626,4627..5418)
                     /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"
     rep_origin      5589..6177
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6078..6097
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatctaa catatcctgg tgtggagtag gggacgctgc
      481 tctgacagag gctcgggggc ctgagctggc tctgtgagct ggggaggagg cagacagcca
      541 ggccttgtct gcaagcagac ctggcagcat tgggctggcc gccccccagg gcctcctctt
      601 catgcccagt gaatgactca ccttggcaca gacacaatgt tcggggtggg cacagtgcct
      661 gcttcccgcc gcaccccagc ccccctcaaa tgccttccga gaagcccatt gagcaggggg
      721 cttgcattgc accccagcct gacagcctgg catcttggga taaaagcagc acagccccct
      781 aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga acaaggctct attcagcctg
      841 tgcccaggaa aggggatcag gggatgccca ggcatggaca gtgggtggca gggggggaga
      901 ggagggctgt ctgcttccca gaagtccaag gacacaaatg ggtgagggga gagctctccc
      961 catagctggg ctgcggccca accccacccc ctcaggctat gccagggggt gttgccaggg
     1021 gcacccgggc atcgccagtc tagcccactc cttcataaag ccctcgcatc ccaggagcga
     1081 gcagagccag agcaggttgg agaggagacg catcacctcc gctgctcgca agctttattg
     1141 cggtagttta tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga
     1201 acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
     1261 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
     1321 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
     1381 cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta taggctagcc
     1441 tcgagctcaa gcttcgaatt caccaccatg tacccatacg atgttcctga ctatgcggac
     1501 tacaaagacg atgacgacaa gaagtacctg gaggaccggg gcgaggtgac ttttgagaag
     1561 atcttctccc agaagctggg gtacctgctt ttccgagact tctgcctgaa gcacctggag
     1621 gaggccaagc ccttggtaga gttctacgag gagatcaaga aatacgagaa gctggagaca
     1681 gaggaggagc gcctggtctg cagccgagag atcttcgcca cgtacatcat gaaggagctg
     1741 ctggcctgct cacatccttt ctcgaagagc gccattgagc acgtccaggg ccatctggtg
     1801 aagaagcagg tgcctccgga tctcttccag ccatatattg aagaaatttg ccagaacctc
     1861 cgaggagacg tgttccagaa attcatcgag agcgataaat tcacacgatt ttgccagtgg
     1921 aagaatgtag agctcaacat ccacggtagt ggcgcaacaa acttctctct gctgaaacaa
     1981 gccggagatg tcgaagagaa tcctggaccg atggtgagca agggcgagga ggataacatg
     2041 gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac
     2101 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     2161 ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc
     2221 atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg
     2281 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     2341 accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc
     2401 ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
     2461 tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg
     2521 aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag
     2581 cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac
     2641 gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc
     2701 atggacgagc tgtacaagta gaccggtatc atcaagctta tcgataatca acctctggat
     2761 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     2821 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     2881 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     2941 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     3001 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     3061 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3121 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc
     3181 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3241 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     3301 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat acctctagag tcgacccggg
     3361 cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga
     3421 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     3481 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     3541 cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc
     3601 gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt taatcattaa
     3661 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     3721 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     3781 cgagcgagcg cgcagcctta attaacctaa ttcactggcc gtcgttttac aacgtcgtga
     3841 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     3901 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     3961 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4021 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4081 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4141 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4201 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4261 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     4321 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4381 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     4441 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4501 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4561 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4621 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4681 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4741 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4801 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4861 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4921 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4981 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5041 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5101 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5161 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5221 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     5281 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     5341 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5401 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     5461 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     5521 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5581 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5641 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5701 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5761 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5821 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5881 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5941 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6001 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6061 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6121 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6181 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6241 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     6301 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
