LOCUS       sequence-336414-        6252 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the expression of His-GBAi W211A in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6252)
  AUTHORS   Leyme A, Marivin A, Maziarz M, DiGiacomo V, Papakonstantinou MP, 
            Patel PP, Blanco-Canosa JB, Walawalkar IA, Rodriguez-Davila G, 
            Dominguez I, Garcia-Marcos M
  TITLE     Specific inhibition of GPCR-independent G protein signaling by a 
            rationally engineered protein.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Nov 28;114(48):E10319-E10328. doi: 
            10.1073/pnas.1707992114. Epub 2017 Nov 13.
  PUBMED    29133411
REFERENCE   2  (bases 1 to 6252)
  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: 
            "10.1073/pnas.1707992114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-11-28- 28"; volume: "114"; issue: "48"; pages: 
            "E10319-E10328"
COMMENT     Sequence Label: pLIC-His-GBAi W211A
FEATURES             Location/Qualifiers
     source          1..6252
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     957..976
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(996..1073)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1279..1298
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1386..1405
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1386..1404
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1405..1429
                     /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)."
     RBS             1444..1466
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1477..1494
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1519..1539
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2574..2591
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2644..2662)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2658..2705
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2829..2848)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3015..3118
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3119..3187,3188..3979)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3337..3356)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4150..4738
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4639..4658
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4892..4909
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4924..5063
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4989..5008)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5149..5171
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5165..5356)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6165..6252,1..995))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
       61 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
      121 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
      181 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
      241 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
      301 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
      361 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
      421 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
      481 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
      541 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
      601 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
      661 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
      721 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
      781 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
      841 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
      901 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
      961 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     1021 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     1081 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     1141 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     1201 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     1261 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     1321 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc
     1381 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     1441 aattttgttt aactttaaga aggagatata catatgcacc atcatcatca tcattcttct
     1501 ggtgtagatc tgggtaccga gaacctgtac ttccaatcca atgcggacgg agagaaagcg
     1561 gccaaagaag tgaagctgct gctgctcggc gctggagaat ctggtaaaag tactattgtg
     1621 aaacagatga aaatcattca tgaggatggc tattccgagg acgagtgtaa gcagtataaa
     1681 gttgtcgtct acagcaatac cattcagtcc atcattgcaa tcataagagc catgggacgg
     1741 ttgaagattg attttgggga agctgccaga gcggatgatg cccgacagtt atttgtttta
     1801 gctggcagtg ctgaggaagg agtcatgact tcagaactag caggcgtgat taaacgttta
     1861 tggcgagatg gcggggtgca ggcatgcttc agcaggtcca gggaatatca gctcattgat
     1921 tctgcttcat attacctaaa tgatttggat agaatatccc agaccaacta cattccaact
     1981 cagcaagatg ttcttcggac gagagtgaag actacaggca ttgtggagac ccacttcacc
     2041 ttcaaggagc tctacttcaa aatgtttgat gtaggtgccc aaagatccga acggaaaaag
     2101 gcgatccact gttttgaggg agtgacagca attatctttt gtgtggctct cagtgattac
     2161 gaccttgttc tggctgagga cgaggaaatg aaccgaatgc atgaaagcat gaaattgttt
     2221 gacagcattt gtaacaacaa atggtttaca gacacttcaa tcattctctt ccttaataag
     2281 aaagaccttt ttgaggaaaa aataaagagg agtccattaa caatctgtta tccagaatac
     2341 acaggttcca atacgtacga agaggcagct gcttacatcc agtgccagtt tgaagatctg
     2401 aaccgaagaa aggacaccaa ggaggtctac actcacttta cctgtgccac tgacaccaaa
     2461 aacgtgcagt ttgtttttga tgctgttaca gatgtcatca ttaaatagcg cattggaagt
     2521 ggataacgga tccgaattcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc
     2581 accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     2641 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     2701 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     2761 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2821 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     2881 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     2941 ccccaaaaaa cttgattagg gtgatggttc acattaacgc ttacaattta ggtggcactt
     3001 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3061 atccgctcat gagacaataa ccctgataaa tgcttcaata atttgaaaaa ggaagagtat
     3121 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3181 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     3241 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     3301 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     3361 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     3421 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     3481 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3541 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3601 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3661 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3721 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3781 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     3841 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3901 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3961 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4021 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4081 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4141 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4201 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     4261 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     4321 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     4381 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     4441 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     4501 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4561 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4621 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4681 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4741 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4801 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4861 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4921 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     4981 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     5041 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     5101 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     5161 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     5221 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     5281 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     5341 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     5401 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     5461 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     5521 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     5581 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     5641 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     5701 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     5761 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     5821 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     5881 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     5941 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     6001 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     6061 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     6121 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     6181 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6241 gtttgcgtat tg
//
