LOCUS       sequence-497011-        5566 bp    DNA     circular SYN 13-JAN-2026
DEFINITION  Vector for protein expression of 2x StrepII-tagged human 
            heterotrimeric G protein GNAO1 in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5566)
  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 5566)
  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-GNAO1
FEATURES             Location/Qualifiers
     source          1..5566
                     /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             1515..1538
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1617..1639)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1784..1802)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1866..1970
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1971..2039,2040..2831)
                     /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(2189..2208)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3002..3590
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3491..3510
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3744..3761
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3834..3911
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3912..4994
                     /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(3931..3950)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5043..5048,5049..5066,5067..5073)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5081..5097
                     /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     5105..5121
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5105..5121
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5117..5566,1..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(5137..5154)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5137..5153)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5146..5168)
                     /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 tgatgggatg tactctgagc gcagaggaga gagccgccct cgagcggagc aaggcgattg
      481 agaaaaacct caaagaggat ggcatcagcg ccgccaaaga cgtgaaatta ctcctgctcg
      541 gggctggaga atcaggaaaa agcaccattg tgaagcagat gaagatcatc catgaagatg
      601 gcttctccgg agaagacgtg aaacagtaca agcctgttgt ctacagcaac actatccagt
      661 ccctggcagc catcgtccgg gccatggaca ctttgggcat cgaatatggt gataaggaga
      721 gaaaggctga cgccaagatg gtgtgtgatg tggtgagtcg gatggaagac accgagccct
      781 tctctgcaga gctgctttct gccatgatgc ggctctgggg cgactcagga atccaagagt
      841 gcttcaaccg gtcccgggag tatcagctca acgactctgc caaatactac ctggacagcc
      901 tggatcggat tggggccgcc gactaccagc ccaccgagca ggacatcctc cgaaccaggg
      961 tcaaaaccac tggcatcgta gaaacccact tcacattcaa gaacctccac ttcaggctgt
     1021 ttgacgtcgg aggccagcga tctgaacgca agaagtggat ccattgcttc gaggacgtca
     1081 cggccatcat tttctgtgtc gcgctcagcg gctatgacca ggtgctccac gaagacgaaa
     1141 ccacgaaccg catgcacgag tctctcatgc tcttcgactc catctgtaac aacaagttct
     1201 tcatcgatac ctccatcatt ctcttcctca acaagaaaga tctctttggc gagaagatca
     1261 agaagtcacc tttgaccatc tgctttcctg aatacacagg ccccaatacc tatgaagacg
     1321 cagccgccta catccaagca caatttgaaa gcaaaaaccg ctcacccaac aaagaaatat
     1381 attgtcacat gacttgtgcc acagacacga ataacatcca ggtggtgttc gacgccgtca
     1441 ccgacatcat cattgccaac aacctccggg gctgcggctt gtactgagtg acgtccggag
     1501 cgagcaccgg tgcagattat aaggatgatg atgataagta actgagtaag cgatcgcaga
     1561 attcatcgtg actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc
     1621 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     1681 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag
     1741 tcacgtagcg atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta
     1801 tttttatagg ttaatgtcat gataataatg gtttcttagc aggtggcact tttcggggaa
     1861 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     1921 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     1981 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2041 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     2101 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2161 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg
     2221 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     2281 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     2341 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     2401 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     2461 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     2521 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     2581 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     2641 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     2701 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     2761 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     2821 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     2881 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     2941 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3001 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3061 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3121 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3181 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3241 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3301 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3361 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3421 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3481 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3541 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3601 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3661 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3721 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     3781 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataaat tccgacacca
     3841 tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
     3901 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
     3961 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
     4021 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
     4081 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
     4141 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
     4201 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
     4261 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
     4321 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
     4381 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
     4441 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
     4501 gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg
     4561 aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca
     4621 tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca
     4681 ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg
     4741 aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg
     4801 ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc
     4861 agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg
     4921 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     4981 aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccccag
     5041 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     5101 cacacaggaa acagctatga ccatgattac ggattcactg gccgtcgttt tacaacgtcg
     5161 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     5221 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     5281 gaatggcgaa tggcgctttg cctggtttcc ggcaccagaa gcggtgccgg aaagctggct
     5341 ggagtgcgat cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg
     5401 ttacgatgcg cccatctaca ccaacgtgac ctatcccatt acggtcaatc cgccgtttgt
     5461 tcccacggag aatccgacgg gttgttactc gctcacattt aatgttgatg aaagctggct
     5521 acaggaaggc cagacgcgaa ttatttttga tggcgttgga attagc
//
