LOCUS       sequence-227774-        7205 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-iBARK(R106A)-p2A-mCherry: Negative control mammalian expression 
            vector for Gaq silencing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7205)
  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 7205)
  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: CMV BARK R106A p2A mCherry
FEATURES             Location/Qualifiers
     source          1..7205
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        140..519
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        520..723
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     674..694
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     768..787
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        768..786
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             803..829
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             830..853
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1286..1342
                     /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             1343..2053
                     /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(1488..1506)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1756..1775
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2060..2648
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2113..2133)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2531..2542)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        complement(2664..2682)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2665..2682)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2702..2719)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2708..2932
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2978..3406
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3065..3084)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3275..3296
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3415..3435)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3420..3749
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3600..3735
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3662..3681
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3815..4609
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3869..3888)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4479..4498
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4783..4904
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4820..4839)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4874..4893
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4953..4969)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4953..4969)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4966..4988)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4977..4993
                     /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)."
     promoter        complement(join(5001..5007,5008..5025,5026..5031))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5046..5067
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5184..5201)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5355..5943)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5435..5454)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6114..6905,6906..6974))
                     /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     6737..6756
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6975..7079)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7154..7173)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
       61 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
      121 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
      181 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      241 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      301 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      361 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      421 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      481 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      541 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      601 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      661 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      721 gctctctggc taactagaga acccactgct tactggctta tcgaaattaa tacgactcac
      781 tatagggaga cccaagctta tgtacccata cgatgttcct gactatgcgg actacaaaga
      841 cgatgacgac aagaagtacc tggaggaccg gggcgaggtg acttttgaga agatcttctc
      901 ccagaagctg gggtacctgc ttttccgaga cttctgcctg aagcacctgg aggaggccaa
      961 gcccttggta gagttctacg aggagatcaa gaaatacgag aagctggaga cagaggagga
     1021 gcgcctggtc tgcagcgcag agatcttcga cacgtacatc atgaaggagc tgctggcctg
     1081 ctcacatcct ttctcgaaga gcgccattga gcacgtccag ggccatctgg tgaagaagca
     1141 ggtgcctccg gatctcttcc agccatatat tgaagaaatt tgccagaacc tccgaggaga
     1201 cgtgttccag aaattcatcg agagcgataa attcacacga ttttgccagt ggaagaatgt
     1261 agagctcaac atccacggta gtggcgcaac aaacttctct ctgctgaaac aagccggaga
     1321 tgtcgaagag aatcctggac cgatggtgag caagggcgag gaggataaca tggccatcat
     1381 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
     1441 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     1501 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
     1561 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     1621 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     1681 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     1741 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga
     1801 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     1861 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
     1921 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta
     1981 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
     2041 gctgtacaag tagtctagaa atcaacctct ggattacaaa atttgtgaaa gattgactgg
     2101 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     2161 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     2221 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     2281 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     2341 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     2401 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     2461 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     2521 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     2581 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     2641 ctccccgcac tagagggccc tattctatag tgtcacctaa atgctagagc tcgctgatca
     2701 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     2761 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     2821 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     2881 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     2941 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     3001 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     3061 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3121 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3181 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3241 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3301 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3361 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     3421 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3481 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3541 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3601 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3661 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     3721 gcttttttgg aggcctaggc ttttgcaaaa agctcccgga gcttgtatat ccattttcgg
     3781 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3841 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3901 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3961 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg
     4021 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     4081 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     4141 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4201 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4261 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4321 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4381 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4441 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4501 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4561 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4621 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4681 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4741 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct
     4801 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4861 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg
     4921 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     4981 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5041 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5101 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5161 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5221 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5281 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5341 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5401 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5461 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5521 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     5581 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5641 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5701 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5761 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     5821 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5881 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5941 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6001 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6061 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6121 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6181 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6241 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6301 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6361 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6421 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6481 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6541 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     6601 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6661 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6721 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6781 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6841 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6901 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6961 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7021 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7081 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
     7141 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     7201 cttgt
//
