LOCUS       sequence-246996-        7621 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Eukaryotic expression of the GB2 subunit of the metabotropic GABA 
            receptor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7621)
  AUTHORS   Masharina A, Reymond L, Maurel D, Umezawa K, Johnsson K
  TITLE     A fluorescent sensor for GABA and synthetic GABA(B) receptor 
            ligands.
  JOURNAL   J Am Chem Soc. 2012 Nov 21;134(46):19026-34. doi: 10.1021/ja306320s.
            Epub 2012 Nov 5.
  PUBMED    23095089
REFERENCE   2  (bases 1 to 7621)
  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: "10.1021/ja306320s";
            journalName: "J Am Chem Soc"; date: "2012-11-21- 21"; volume: "134";
            issue: "46"; pages: "19026-34"
COMMENT     Sequence Label: pRK5-GB2
FEATURES             Location/Qualifiers
     source          1..7621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             1013..1036
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3921..3930
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    3934..4068
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3971..3990)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4025..4044
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4137..4494
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4137..4328
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4160..4180)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4345..4480
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4514..4531)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4514..4530)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4523..4545)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4743..5198
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4830..4849)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5040..5061
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5215..5234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5334..5356
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5394..5412)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5480..5584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5585..5653,5654..6445)
                     /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(5803..5822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6616..7204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7105..7124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7358..7375
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7492..7513
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7528..7533,7534..7551,7552..7558)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7566..7582
                     /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     7571..7593
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7590..7606
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7590..7606
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcctttcc taaaatggtc cttctgttga tcctgtcagt
      961 cctacttctg aaagaagatg tacgagggag tgcacagtcc acgcgaccgg tagattataa
     1021 agatgacgat gacaaaacgc gttggacgcg gggcgccccc cggccgccgc ccagcagccc
     1081 gccgctctcc atcatgggcc tcatgccgct caccaaggag gtggccaagg gcagcatcgg
     1141 gcgcggcgtg ctccccgccg tggagctagc catcgagcag atccgcaacg agtcactcct
     1201 gcgcccctac ttcctggacc tgcgactcta tgacaccgag tgtgacaatg caaagggact
     1261 gaaagccttc tatgacgcaa taaagtatgg gccgaaccat ttgatggtgt ttggaggcgt
     1321 ctgtccgtct gtcacatcta ttatcgcgga gtccctccaa ggctggaatc tggtgcagct
     1381 ttccttcgcc gccaccacgc ctgttcttgc ggataagaag aagtacccgt atttcttccg
     1441 gacggtgccg tcagacaacg cggtgaaccc cgccatcctg aagctcctga agcacttccg
     1501 ctggcggcgt gtgggcacac tcacgcagga cgtgcagcgc ttctccgagg tgaggaatga
     1561 cctgactggg gttctgtatg gggaagatat tgagatctca gacacagaga gcttctccaa
     1621 tgatccctgc accagcgtca aaaagctcaa ggggaatgac gtgcggatca tccttggcca
     1681 gtttgaccag aatatggcag caaaagtctt ctgttgtgcc ttcgaggaga gcatgtttgg
     1741 cagcaagtac cagtggatca tcccgggatg gtacgagcct gcgtggtggg agcaggtgca
     1801 tgtggaggcc aattcctcac gctgcctgcg cagaagcctc ctggctgcca tggaaggtta
     1861 catcggagtg gactttgagc ccctgagctc caaacaaatc aagaccatct cagggaagac
     1921 tccacagcag tatgaaagag agtacaacac caaacgttca ggcgtggggc ccagcaagtt
     1981 ccatgggtac gcctacgatg ggatctgggt catcgccaag accctacaga gggccatgga
     2041 gacactgcat gccagtagca ggcaccagcg gatccaggac ttcaactaca cagaccacac
     2101 gctgggcaaa atcatcctca atgccatgaa cgagaccaac ttcttcgggg tcacgggtca
     2161 agttgtgttc cggaacgggg agagaatggg aaccattaaa tttactcaat ttcaagacag
     2221 cagagaggtg aaggtcggcg aatacaacgc ggtggctgac acactggaga tcatcaatga
     2281 caccataagg ttccaggggt ccgagccacc caaggacaag accatcattc tggagcagct
     2341 tcggaagatc tcgcttccac tgtatagcat cctgtccgct ctcaccatcc tcggcatgat
     2401 catggccagc gccttcctct tcttcaacat caagaaccgg aaccaaaagc tgattaagat
     2461 gtcaagcccc tacatgaaca acctcatcat cctgggagga atgctgtcct atgcatccat
     2521 cttcctcttt ggcctcgatg ggtccttcgt ctcagaaaag acctttgaaa cactctgcac
     2581 ggtccggacc tggattctca ccgtgggcta cacaactgcc tttggggcca tgtttgcaaa
     2641 gacctggagg gtccatgcca tcttcaaaaa tgtgaagatg aagaagaaga tcatcaaaga
     2701 ccagaagctg cttgtgattg tggggggcat gctgctcatc gacctgtgca tcctgatctg
     2761 ttggcaggct gtggaccccc tgcggaggac agtagagagg tacagcatgg agccggaccc
     2821 agcaggccgg gacatctcca tccgcccatt gctggaacac tgcgaaaaca cccacatgac
     2881 catctggctt ggcattgtct acgcctacaa ggggctcctc atgctattcg gttgtttctt
     2941 ggcatgggaa acccgcaatg tgagcatccc tgccctcaac gacagcaagt acatcggcat
     3001 gagtgtgtac aatgtgggga tcatgtgcat catcggggct gctgtctcct tcctgacgcg
     3061 tgaccagccc aacgtgcagt tctgcatcgt ggccctggtc atcatcttct gcagcaccat
     3121 cactctctgc ctggtgtttg tgccaaagct catcactctg aggacaaacc ctgacgcagc
     3181 cactcagaac aggcggttcc agttcacaca gaaccagaag aaagaagatt cgaagacctc
     3241 cacttcagtc accagcgtga accaggcgag cacgtcacgc ctggagggac tgcagtcaga
     3301 aaaccaccgc cttcgaatga agatcacaga gctggacaaa gacttggaag aagtcaccat
     3361 gcagctacaa gacacaccag agaagaccac atacatcaaa cagaatcact accaagagct
     3421 caacgcggcc gctagcttgg gcaacttcac agagagcaca gatggaggaa aggccattct
     3481 aaaaaatcac ctcgatcaaa acccccagct ccagtggaac acgacagagc cctcaagaac
     3541 atgcaaagac cccatagaag acatcaactc cccggagcac atccagcgcc ggctgtcgct
     3601 ccagctcccc atccttcacc acgcctacct cccatccatc ggaggcgtgg atgccagctg
     3661 cgtcagcccc tgtgtcagcc ctaccgccag ccctcgccac agacacgtac caccctcctt
     3721 ccgagtcatg gtctcgggcc tgtaggggtg ggaggcctgg ggccggggcc tccccgggac
     3781 agcaccatgc tgggccaagg cgcctgccac aggcacactg acggcggcga gaagctgggc
     3841 accatgctgc ctctccagac tgctggaatg gcgctcaggc agagcgggac tcggcaccga
     3901 cctcgacctg cagaagcttg gccgccatgg cccaacttgt ttattgcagc ttataatggt
     3961 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4021 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcgg gaattaattc
     4081 ggcgcagcac catggcctga aataacctct gaaagaggaa cttggttagg taccttctga
     4141 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     4201 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     4261 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4321 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4381 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     4441 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctg
     4501 ttaacagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     4561 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     4621 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc
     4681 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     4741 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4801 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4861 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4921 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     4981 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     5041 tggactcttg ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt
     5101 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     5161 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     5221 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     5281 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     5341 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     5401 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     5461 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5521 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5581 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5641 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5701 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5761 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5821 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5881 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5941 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     6001 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     6061 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     6121 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     6181 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     6241 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     6301 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     6361 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     6421 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6481 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6541 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6601 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6661 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6721 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     6781 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6841 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6901 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6961 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     7021 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     7081 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     7141 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     7201 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     7261 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     7321 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     7381 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     7441 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     7501 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     7561 tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga ttacgaatta
     7621 a
//
