LOCUS       sequence-229475-        7207 bp    DNA     circular SYN 15-APR-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 7207)
  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 7207)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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..7207
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             898..924
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             925..948
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1381..1437
                     /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             1438..2148
                     /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(1583..1601)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1851..1870
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2155..2743
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2208..2228)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2626..2637)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     promoter        complement(2759..2777)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2760..2777)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2797..2814)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2803..3027
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3073..3501
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3160..3179)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3370..3391
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3510..3530)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3515..3845
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3696..3831
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3758..3777
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3912..4706
                     /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(3966..3985)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4576..4595
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4880..5001
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4917..4936)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4971..4990
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5050..5066)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5050..5066)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5063..5085)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5074..5090
                     /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(5098..5104,5105..5122,5123..5128))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5143..5164
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5281..5298)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5452..6040)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5532..5551)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6211..7002,7003..7071))
                     /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     6834..6853
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7072..7176)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttatgtac
      901 ccatacgatg ttcctgacta tgcggactac aaagacgatg acgacaagaa gtacctggag
      961 gaccggggcg aggtgacttt tgagaagatc ttctcccaga agctggggta cctgcttttc
     1021 cgagacttct gcctgaagca cctggaggag gccaagccct tggtagagtt ctacgaggag
     1081 atcaagaaat acgagaagct ggagacagag gaggagcgcc tggtctgcag cgcagagatc
     1141 ttcgacacgt acatcatgaa ggagctgctg gcctgctcac atcctttctc gaagagcgcc
     1201 attgagcacg tccagggcca tctggtgaag aagcaggtgc ctccggatct cttccagcca
     1261 tatattgaag aaatttgcca gaacctccga ggagacgtgt tccagaaatt catcgagagc
     1321 gataaattca cacgattttg ccagtggaag aatgtagagc tcaacatcca cggtagtggc
     1381 gcaacaaact tctctctgct gaaacaagcc ggagatgtcg aagagaatcc tggaccgatg
     1441 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     1501 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     1561 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     1621 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     1681 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     1741 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     1801 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     1861 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     1921 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     1981 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     2041 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     2101 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtagtc tagaaatcaa
     2161 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     2221 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     2281 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     2341 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     2401 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     2461 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     2521 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctat
     2581 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     2641 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     2701 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcactagag ggccctattc
     2761 tatagtgtca cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc
     2821 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2881 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2941 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3001 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     3061 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3121 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3181 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggc atccctttag
     3241 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3301 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3361 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3421 cttttgattt ataagggatt ttggggattt cggcctattg gttaaaaaat gagctgattt
     3481 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3541 cccaggctcc ccaggcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3601 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3661 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3721 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3781 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3841 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     3901 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     3961 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4021 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4081 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4141 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4201 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4261 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     4321 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     4381 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     4441 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     4501 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     4561 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     4621 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     4681 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     4741 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     4801 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     4861 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     4921 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4981 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     5041 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5101 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5161 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5221 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5281 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5341 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5401 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5461 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5521 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5581 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5641 ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5701 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5761 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5821 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5881 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5941 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6001 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6061 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6121 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6181 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6241 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6301 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6361 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6421 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6481 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6541 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6601 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6661 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6721 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6781 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6841 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6901 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6961 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7021 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7081 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7141 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7201 tgacgtc
//
