LOCUS       sequence-336408-        6252 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the expression of His-GBAi 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
FEATURES             Location/Qualifiers
     source          1..6252
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1287..1306
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1394..1413
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1394..1412
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1413..1437
                     /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             1452..1474
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1485..1502
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1527..1547
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2582..2599
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2652..2670)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2666..2713
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2837..2856)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3023..3126
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3127..3195,3196..3987)
                     /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(3345..3364)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4158..4746
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4647..4666
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4900..4917
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4932..5071
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4997..5016)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5157..5179
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5173..5364)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6173..6252,1..1003))
                     /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 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1081 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1141 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1201 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     1261 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     1321 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgatctc
     1381 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1441 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaccat catcatcatc
     1501 attcttctgg tgtagatctg ggtaccgaga acctgtactt ccaatccaat gcggacggag
     1561 agaaagcggc caaagaagtg aagctgctgc tgctcggcgc tggagaatct ggtaaaagta
     1621 ctattgtgaa acagatgaaa atcattcatg aggatggcta ttccgaggac gagtgtaagc
     1681 agtataaagt tgtcgtctac agcaatacca ttcagtccat cattgcaatc ataagagcca
     1741 tgggacggtt gaagattgat tttggggaag ctgccagagc ggatgatgcc cgacagttat
     1801 ttgttttagc tggcagtgct gaggaaggag tcatgacttc agaactagca ggcgtgatta
     1861 aacgtttatg gcgagatggc ggggtgcagg catgcttcag caggtccagg gaatatcagc
     1921 tcattgattc tgcttcatat tacctaaatg atttggatag aatatcccag accaactaca
     1981 ttccaactca gcaagatgtt cttcggacga gagtgaagac tacaggcatt gtggagaccc
     2041 acttcacctt caaggagctc tacttcaaaa tgtttgatgt aggtgcccaa agatccgaac
     2101 ggaaaaagtg gatccactgt tttgagggag tgacagcaat tatcttttgt gtggctctca
     2161 gtgattacga ccttgttctg gctgaggacg aggaaatgaa ccgaatgcat gaaagcatga
     2221 aattgtttga cagcatttgt aacaacaaat ggtttacaga cacttcaatc attctcttcc
     2281 ttaataagaa agaccttttt gaggaaaaaa taaagaggag tccattaaca atctgttatc
     2341 cagaatacac aggttccaat acgtacgaag aggcagctgc ttacatccag tgccagtttg
     2401 aagatctgaa ccgaagaaag gacaccaagg aggtctacac tcactttacc tgtgccactg
     2461 acaccaaaaa cgtgcagttt gtttttgatg ctgttacaga tgtcatcatt aaatagcgca
     2521 ttggaagtgg ataacggatc cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga
     2581 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     2641 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     2701 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     2761 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2821 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2881 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2941 cacctcgacc ccaaaaaact tgattagggt gatggttcac attaacgctt acaatttagg
     3001 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     3061 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat ttgaaaaagg
     3121 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     3181 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     3241 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     3301 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     3361 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     3421 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     3481 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     3541 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     3601 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     3661 acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     3721 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     3781 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     3841 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     3901 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     3961 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     4021 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     4081 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4141 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     4201 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4261 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4321 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4381 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4441 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4501 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4561 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4621 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4681 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4741 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4801 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4861 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4921 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4981 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     5041 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     5101 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     5161 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     5221 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     5281 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     5341 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     5401 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     5461 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     5521 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     5581 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     5641 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     5701 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     5761 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     5821 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     5881 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     5941 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     6001 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     6061 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     6121 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     6181 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     6241 gagaggcggt tt
//
