LOCUS       sequence-227772-        7205 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-iBARK(D110A)-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 D110A p2A mCherry
FEATURES             Location/Qualifiers
     source          1..7205
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        57..436
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        437..640
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     591..611
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     685..704
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        685..703
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             720..746
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             747..770
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1203..1259
                     /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             1260..1970
                     /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(1405..1423)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1673..1692
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    1977..2565
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2030..2050)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2448..2459)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        complement(2581..2599)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2582..2599)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2619..2636)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2625..2849
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2895..3323
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2982..3001)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3192..3213
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3332..3352)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3337..3666
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3517..3652
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3579..3598
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3732..4526
                     /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(3786..3805)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4396..4415
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4700..4821
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4737..4756)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4791..4810
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4870..4886)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4870..4886)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4883..4905)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4894..4910
                     /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(4918..4924,4925..4942,4943..4948))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4963..4984
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5101..5118)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5272..5860)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5352..5371)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6031..6822,6823..6891))
                     /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     6654..6673
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6892..6996)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7071..7090)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
       61 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      121 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      181 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      241 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      301 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      361 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      421 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      481 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      541 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      601 cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc
      661 cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc aagcttatgt
      721 acccatacga tgttcctgac tatgcggact acaaagacga tgacgacaag aagtacctgg
      781 aggaccgggg cgaggtgact tttgagaaga tcttctccca gaagctgggg tacctgcttt
      841 tccgagactt ctgcctgaag cacctggagg aggccaagcc cttggtagag ttctacgagg
      901 agatcaagaa atacgagaag ctggagacag aggaggagcg cctggtctgc agccgagaga
      961 tcttcgccac gtacatcatg aaggagctgc tggcctgctc acatcctttc tcgaagagcg
     1021 ccattgagca cgtccagggc catctggtga agaagcaggt gcctccggat ctcttccagc
     1081 catatattga agaaatttgc cagaacctcc gaggagacgt gttccagaaa ttcatcgaga
     1141 gcgataaatt cacacgattt tgccagtgga agaatgtaga gctcaacatc cacggtagtg
     1201 gcgcaacaaa cttctctctg ctgaaacaag ccggagatgt cgaagagaat cctggaccga
     1261 tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg
     1321 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     1381 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct
     1441 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc
     1501 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg
     1561 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
     1621 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
     1681 tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg
     1741 ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg
     1801 aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca
     1861 acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac
     1921 gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtag tctagaaatc
     1981 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     2041 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     2101 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     2161 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     2221 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     2281 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     2341 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     2401 atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     2461 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     2521 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcactag agggccctat
     2581 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     2641 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2701 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2761 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     2821 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     2881 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     2941 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3001 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3061 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3121 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3181 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3241 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3301 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3361 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3421 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3481 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3541 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3601 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3661 tgcaaaaagc tcccggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     3721 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3781 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     3841 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     3901 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     3961 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4021 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4081 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4141 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4201 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4261 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4321 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4381 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4441 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4501 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4561 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4621 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4681 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     4741 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4801 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     4861 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4921 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4981 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5041 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5101 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5161 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5221 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5281 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5341 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5401 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5461 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5521 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5581 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5641 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5701 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5761 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5821 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5881 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5941 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6001 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6061 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6121 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6181 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6241 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6301 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6361 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6421 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6481 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6541 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6601 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6661 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6721 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6781 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6841 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6901 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6961 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7021 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     7081 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     7141 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     7201 aatct
//
