LOCUS       sequence-497016-        5557 bp    DNA     circular SYN 13-JAN-2026
DEFINITION  Vector for protein expression of 2x StrepII-tagged human 
            heterotrimeric G protein GNAI1 in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5557)
  AUTHORS   Gookin TE, Chakravorty D, Assmann SM
  TITLE     Influence of expression and purification protocols on Galpha 
            biochemical activity: kinetics of plant and mammalian G protein 
            cycles.
  JOURNAL   Front Mol Biosci. 2025 Apr 7;12:1513660. doi: 
            10.3389/fmolb.2025.1513660. eCollection 2025.
  PUBMED    40260404
REFERENCE   2  (bases 1 to 5557)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 13, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Front Mol 
            Biosci."; date: "2025-04-7"; volume: "12:1513660. doi"; pages: " 
            10.3389/fmolb.2025.1513660. eCollection 2025"
COMMENT     Sequence Label: pSTTa-GNAI1
FEATURES             Location/Qualifiers
     source          1..5557
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(180..185,186..201,202..208)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    216..232
                     /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)."
     CDS             273..296
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             330..353
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             363..380
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             387..407
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1506..1529
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1608..1630)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1775..1793)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1857..1961
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1962..2030,2031..2822)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2180..2199)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2993..3581
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3482..3501
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3735..3752
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3825..3902
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3903..4985
                     /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"
     primer_bind     complement(3922..3941)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5034..5039,5040..5057,5058..5064)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5072..5088
                     /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     5096..5112
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5096..5112
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5108..5557,1..78)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5128..5145)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5128..5144)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5137..5159)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta
       61 tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct
      121 ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt
      181 tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac aatttcacac
      241 aggaaacagt attcatgagc gctagcagcg cgtggagcca cccgcagttc gaaaagagcg
      301 gtggctcagg taccagtggc ggttcagcct ggagccatcc gcaattcgag aaggtcggca
      361 gcctggttcc gcgtggttct gctggtgaaa acctgtactt ccagggtggt tcaggatcca
      421 tgggctgcac gctgagcgcc gaggacaagg cggcggtgga gcggagtaag atgatcgacc
      481 gcaacctccg tgaggacggc gagaaggcgg cgcgcgaggt caagctgctg ctgctcggtg
      541 ctggtgaatc tggtaaaagt acaattgtga agcagatgaa aattatccat gaagctggtt
      601 attcagaaga ggagtgtaaa caatacaaag cagtggtcta cagtaacacc atccagtcaa
      661 ttattgctat cattagggct atggggaggt tgaagataga ctttggtgac tcagcccggg
      721 cggatgatgc acgccaactc tttgtgctag ctggagctgc tgaagaaggc tttatgactg
      781 cagaacttgc tggagttata aagagattgt ggaaagatag tggtgtacaa gcctgtttca
      841 acagatcccg agagtaccag cttaatgatt ctgcagcata ctatttgaat gacttggaca
      901 gaatagctca accaaattac atcccgactc aacaagatgt tctcagaact agagtgaaaa
      961 ctacaggaat tgttgaaacc cattttactt tcaaagatct tcattttaaa atgtttgatg
     1021 tgggaggtca gagatctgag cggaagaagt ggattcattg cttcgaagga gtgacggcga
     1081 tcatcttctg tgtagcactg agtgactacg acctggttct agctgaagat gaagaaatga
     1141 accgaatgca tgaaagcatg aaattgtttg acagcatatg taacaacaag tggtttacag
     1201 atacatccat tatacttttt ctaaacaaga aggatctctt tgaagaaaaa atcaaaaaga
     1261 gccctctcac tatatgctat ccagaatatg caggatcaaa cacatatgaa gaggcagctg
     1321 catatattca atgtcagttt gaagacctca ataaaagaaa ggacacaaag gaaatataca
     1381 cccacttcac atgtgccaca gatactaaga atgtgcagtt tgtttttgat gctgtaacag
     1441 atgtcatcat aaaaaataat ctaaaagatt gtggtctctt ttaatccgga gcgagcaccg
     1501 gtgcagatta taaggatgat gatgataagt aactgagtaa gcgatcgcag aattcatcgt
     1561 gactgactga cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     1621 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     1681 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     1741 gatagcggag tgtataattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     1801 gttaatgtca tgataataat ggtttcttag caggtggcac ttttcgggga aatgtgcgcg
     1861 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1921 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1981 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     2041 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     2101 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     2161 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     2221 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2281 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2341 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2401 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2461 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     2521 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2581 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2641 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2701 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2761 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     2821 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     2881 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2941 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3001 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3061 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3121 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3181 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3241 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3301 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3361 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3421 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3481 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3541 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3601 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3661 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3721 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3781 ttctccttac gcatctgtgc ggtatttcac accgcataaa ttccgacacc atcgaatggc
     3841 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     3901 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     3961 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     4021 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     4081 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     4141 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     4201 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     4261 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     4321 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     4381 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     4441 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     4501 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     4561 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     4621 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     4681 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     4741 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     4801 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     4861 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     4921 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     4981 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacccca ggctttacac
     5041 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     5101 aacagctatg accatgatta cggattcact ggccgtcgtt ttacaacgtc gtgactggga
     5161 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     5221 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     5281 atggcgcttt gcctggtttc cggcaccaga agcggtgccg gaaagctggc tggagtgcga
     5341 tcttcctgag gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg gttacgatgc
     5401 gcccatctac accaacgtga cctatcccat tacggtcaat ccgccgtttg ttcccacgga
     5461 gaatccgacg ggttgttact cgctcacatt taatgttgat gaaagctggc tacaggaagg
     5521 ccagacgcga attatttttg atggcgttgg aattagc
//
