LOCUS       sequence-215924-        7785 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of GNAO1_G-alpha.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7785)
  AUTHORS   Teyra J, Kelil A, Jain S, Helmy M, Jajodia R, Hooda Y, Gu J, D'Cruz 
            AA, Nicholson SE, Min J, Sudol M, Kim PM, Bader GD, Sidhu SS
  TITLE     Large-scale survey and database of high affinity ligands for peptide
            recognition modules.
  JOURNAL   Mol Syst Biol. 2020 Dec;16(12):e9310. doi: 10.15252/msb.20199310.
  PUBMED    33438817
REFERENCE   2  (bases 1 to 7785)
  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"; journalName: "Mol Syst 
            Biol."; date: "2020-12"; volume: "16(12):e9310. doi"; pages: " 
            10.15252/msb.20199310"
COMMENT     Sequence Label: pHH0103_GNAO1_G-alpha
FEATURES             Location/Qualifiers
     source          1..7785
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             867..884
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      1980..2027
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3620..4075)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3757..3778)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3969..3988
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4117..4139)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4225..4365
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4280..4299
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4380..4397)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4551..5138)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5309..6100,6101..6169))
                     /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     5932..5951
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6170..6274)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6342..6360
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6383..6460
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             6461..7543
                     /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(6480..6499)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc
      901 cggccggctg caccctgagt gctgaagaac gtgcggccct ggaacgctcc aaagcaatcg
      961 agaaaaacct gaaagaagac ggcattagcg cagctaaaga tgttaaactg ctgctgctgg
     1021 gcgccggtga aagcggtaaa tctaccatcg tcaaacagat gaaaattatc catgaagatg
     1081 gctttagcgg tgaagacgtg aaacagtata aaccggtggt ttactcaaat accatccaat
     1141 cgctggcggc cattgttcgt gcgatggata cgctgggcat tgaatacggt gataaagaac
     1201 gtaaagctga cgcgaaaatg gtctgcgatg tcgtgagtcg catggaagat accgaaccgt
     1261 ttagcgccga actgctgtct gcaatgatgc gtctgtgggg cgacagcggt atccaggaat
     1321 gtttcaaccg ttctcgcgaa taccaactga atgattcagc gaaatattac ctggattcgc
     1381 tggaccgcat tggcgcagct gactatcagc cgaccgaaca agatatcctg cgtacgcgcg
     1441 tgaaaaccac gggtatcgtt gaaacccatt tcacgttcaa aaacctgcac tttcgtctgt
     1501 tcgacgttgg cggtcagcgt agcgaacgca aaaaatggat tcattgcttt gaagatgtca
     1561 ccgctattat cttctgtgtg gcgctgtctg gttatgacca agtgctgcat gaagatgaaa
     1621 ccacgaatcg catgcacgaa agtctgatgc tgtttgactc catctgcaac aacaaatttt
     1681 tcatcgatac ctcaatcatc ctgtttctga acaaaaaaga cctgtttggc gaaaaaatca
     1741 aaaaatcgcc gctgacgatt tgttttccgg aatataccgg tccgaatacg tacgaagatg
     1801 cggcggcata tattcaggcc caattcgaaa gtaaaaaccg ttccccgaat aaagaaatct
     1861 actgccacat gacctgtgcg accgatacga acaatattca ggttgtcttc gatgccgtta
     1921 cggacattat cattgcaaac aatctgcgcg gctgtggtct gtatgcggcc gcataataaa
     1981 ccgatacaat taaaggctcc ttttggagcc ttttttttgg agattttcaa cgtgaaaaaa
     2041 ttattattcg caattccttt agttgttcct ttctattctc actccgctga aactgttgaa
     2101 agttgtttag caaaacccca tacagaaaat tcatttacta acgtctggaa agacgacaaa
     2161 actttagatc gttacgctaa ctatgagggc tgtctgtgga atgctacagg cgttgtagtt
     2221 tgtactggtg acgaaactca gtgtctagct agagtggcgg tggctctggt tccggtgatt
     2281 ttgattatga aaagatggca aacgctaata agggggctat gaccgaaaat gccgatgaaa
     2341 acgcgctaca gtctgacgct aaaggcaaac ttgattctgt cgctactgat tacggtgctg
     2401 ctatcgatgg tttcattggt gacgtttccg gccttgctaa tggtaatggt gctactggtg
     2461 attttgctgg ctctaattcc caaatggctc aagtcggtga cggtgataat tcacctttaa
     2521 tgaataattt ccgtcaatat ttaccttccc tccctcaatc ggttgaatgt cgcccttttg
     2581 tctttggcgc tggtaaacca tatgaatttt ctattgattg tgacaaaata aacttattcc
     2641 gtggtgtctt tgcgtttctt ttatatgttg ccacctttat gtatgtattt tctacgtttg
     2701 ctaacatact gcgtaataag gagtcttaat catgccagtt cttttggcta gcgccgccct
     2761 ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg
     2821 aatggaagcc ggcggcacct cgctaacgga ttcaccactc caagaattgg agccaatcaa
     2881 ttcttgcgga gaactgtgaa tgcgcaaacc aacccttggc agaacatatc catcgcgtcc
     2941 gccatctcca gcagccgcac gcggcgcatc tcgggcagcg ttgggtcctg gccacgggtg
     3001 cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt
     3061 tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct
     3121 gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg
     3181 cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta
     3241 ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt
     3301 ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg
     3361 gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt
     3421 acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc
     3481 gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac
     3541 gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag
     3601 ctttaccgca ggatccgcga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     3661 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3721 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3781 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3841 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3901 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3961 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     4021 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcgcgg
     4081 atcctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     4141 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     4201 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     4261 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     4321 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc
     4381 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4441 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4501 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4561 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4621 acaggactat aaagatacca gggtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4681 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4741 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4801 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4861 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4921 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4981 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5041 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5101 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5161 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5221 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5281 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5341 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5401 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5461 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5521 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5581 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt
     5641 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5701 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5761 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5821 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5881 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg
     5941 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     6001 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     6061 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     6121 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6181 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6241 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6301 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     6361 cctttcgtct tcaagaattc ccgacaccat cgaatggtgc aaaacctttc gcggtatggc
     6421 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     6481 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     6541 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     6601 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     6661 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     6721 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     6781 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     6841 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     6901 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     6961 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     7021 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     7081 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     7141 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     7201 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     7261 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     7321 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     7381 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     7441 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     7501 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     7561 taagttagct cactcattag gcacaattct catgtttgac agcttatcat cgactgcacg
     7621 gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc
     7681 gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt
     7741 gcgccgacat cataacggtt ctggcaaata ttctgaaatg agctg
//
