LOCUS       sequence-215195-        7773 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression and purification of GNAT1_G-alpha.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7773)
  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 7773)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_GNAT1_G-alpha
FEATURES             Location/Qualifiers
     source          1..7773
                     /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      1968..2015
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3608..4063)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3745..3766)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3957..3976
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4105..4127)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4213..4353
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4268..4287
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4368..4385)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4539..5126)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5297..6088,6089..6157))
                     /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     5920..5939
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6158..6262)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6330..6348
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6371..6448
                     /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             6449..7531
                     /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(6468..6487)
                     /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 cggccggtgc tggtgcaagt gccgaagaaa aacatagccg tgaactggaa aaaaaactga
      961 aagaagatgc cgaaaaagac gcacgtaccg ttaaactgct gctgttaggt gccggtgaaa
     1021 gcggtaaaag caccattgtt aaacaaatga agatcatcca ccaggatggt tatagcctgg
     1081 aagaatgtct ggaattcatt gccattatct atggtaatac cctgcagagc attctggcaa
     1141 ttgttcgtgc aatgaccacc ctgaacattc agtatggtga tagcgcacgt caggatgatg
     1201 cacgtaaact gatgcatatg gcagatacca ttgaagaggg caccatgcct aaagaaatga
     1261 gcgatattat tcagcgcctg tggaaagata gcggtattca ggcatgtttt gaacgtgcaa
     1321 gcgaatatca gctgaatgat agtgcaggtt attatctgag cgatctggaa cgtctggtta
     1381 caccgggtta tgttccgacc gaacaggatg ttctgcgtag ccgtgttaaa accaccggta
     1441 ttattgaaac ccagttcagc ttcaaagatc tgaattttcg catgtttgat gttggtggtc
     1501 agcgtagcga acgtaaaaaa tggattcatt gttttgaggg cgtgacctgc attatcttta
     1561 ttgcagcact gagcgcatat gatatggttc tggttgaaga tgatgaagtg aatcgtatgc
     1621 atgaaagcct gcacctgttt aatagcattt gcaatcatcg ttattttgcc accacctcca
     1681 ttgttctgtt tctgaacaaa aaggatgtgt tttttgagaa aatcaaaaaa gcccacctga
     1741 gcatctgctt tccggattat gatggtccga atacctatga agatgcgggt aattacatca
     1801 aggttcagtt tctggaactg aatatgcgtc gtgatgtgaa agaaatctat agccatatga
     1861 cctgtgcaac cgatacccag aatgtgaaat ttgtttttga tgccgtgacc gatatcatta
     1921 tcaaagaaaa tctgaaggat tgcggtctgt ttgcggccgc ataataaacc gatacaatta
     1981 aaggctcctt ttggagcctt ttttttggag attttcaacg tgaaaaaatt attattcgca
     2041 attcctttag ttgttccttt ctattctcac tccgctgaaa ctgttgaaag ttgtttagca
     2101 aaaccccata cagaaaattc atttactaac gtctggaaag acgacaaaac tttagatcgt
     2161 tacgctaact atgagggctg tctgtggaat gctacaggcg ttgtagtttg tactggtgac
     2221 gaaactcagt gtctagctag agtggcggtg gctctggttc cggtgatttt gattatgaaa
     2281 agatggcaaa cgctaataag ggggctatga ccgaaaatgc cgatgaaaac gcgctacagt
     2341 ctgacgctaa aggcaaactt gattctgtcg ctactgatta cggtgctgct atcgatggtt
     2401 tcattggtga cgtttccggc cttgctaatg gtaatggtgc tactggtgat tttgctggct
     2461 ctaattccca aatggctcaa gtcggtgacg gtgataattc acctttaatg aataatttcc
     2521 gtcaatattt accttccctc cctcaatcgg ttgaatgtcg cccttttgtc tttggcgctg
     2581 gtaaaccata tgaattttct attgattgtg acaaaataaa cttattccgt ggtgtctttg
     2641 cgtttctttt atatgttgcc acctttatgt atgtattttc tacgtttgct aacatactgc
     2701 gtaataagga gtcttaatca tgccagttct tttggctagc gccgccctat accttgtctg
     2761 cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg
     2821 cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt cttgcggaga
     2881 actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc catctccagc
     2941 agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg
     3001 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     3061 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca
     3121 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg
     3181 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     3241 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
     3301 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
     3361 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
     3421 agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg
     3481 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg
     3541 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagg
     3601 atccgcgaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     3661 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     3721 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     3781 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     3841 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     3901 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     3961 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     4021 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcgcggat cctgcctcgc
     4081 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     4141 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     4201 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     4261 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     4321 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     4381 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4441 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4501 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4561 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4621 agataccagg gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4681 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4741 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4801 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4861 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4921 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     4981 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5041 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5101 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5161 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5221 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5281 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5341 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5401 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5461 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5521 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5581 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     5641 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5701 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5761 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5821 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5881 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     5941 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6001 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6061 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6121 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6181 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6241 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     6301 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     6361 aagaattccc gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc
     6421 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     6481 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     6541 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     6601 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     6661 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     6721 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     6781 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     6841 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     6901 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     6961 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     7021 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     7081 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     7141 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     7201 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     7261 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     7321 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     7381 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     7441 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     7501 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     7561 ctcattaggc acaattctca tgtttgacag cttatcatcg actgcacggt gcaccaatgc
     7621 ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc
     7681 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca
     7741 taacggttct ggcaaatatt ctgaaatgag ctg
//
