LOCUS       sequence-213885-        7005 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of GIGYF1_GYF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7005)
  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 7005)
  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_GIGYF1_GYF
FEATURES             Location/Qualifiers
     source          1..7005
                     /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      1200..1247
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2840..3295)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2977..2998)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3189..3208
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3337..3359)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3445..3585
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3500..3519
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3600..3617)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3771..4358)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4529..5320,5321..5389))
                     /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     5152..5171
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5390..5494)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5562..5580
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5603..5680
                     /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             5681..6763
                     /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(5700..5719)
                     /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 cggcccagtt caccgcagcg atgcagactc agggcctgcg tcattctgct gcggcaaccg
      961 ccctgccgct gtctcacggt gcagcgcgta aatggttcta taaggatccg cagggcgaaa
     1021 ttcaaggccc attcaccacg caggagatgg ctgaatggtt tcaggctggt tactttagca
     1081 tgtctctgct ggtgaaacgt ggttgcgacg aaggtttcca acctctgggc gaagtaatca
     1141 aaatgtgggg tcgcgttccg ttcgcgccgg gtccgtcccc gccggcggcc gcataataaa
     1201 ccgatacaat taaaggctcc ttttggagcc ttttttttgg agattttcaa cgtgaaaaaa
     1261 ttattattcg caattccttt agttgttcct ttctattctc actccgctga aactgttgaa
     1321 agttgtttag caaaacccca tacagaaaat tcatttacta acgtctggaa agacgacaaa
     1381 actttagatc gttacgctaa ctatgagggc tgtctgtgga atgctacagg cgttgtagtt
     1441 tgtactggtg acgaaactca gtgtctagct agagtggcgg tggctctggt tccggtgatt
     1501 ttgattatga aaagatggca aacgctaata agggggctat gaccgaaaat gccgatgaaa
     1561 acgcgctaca gtctgacgct aaaggcaaac ttgattctgt cgctactgat tacggtgctg
     1621 ctatcgatgg tttcattggt gacgtttccg gccttgctaa tggtaatggt gctactggtg
     1681 attttgctgg ctctaattcc caaatggctc aagtcggtga cggtgataat tcacctttaa
     1741 tgaataattt ccgtcaatat ttaccttccc tccctcaatc ggttgaatgt cgcccttttg
     1801 tctttggcgc tggtaaacca tatgaatttt ctattgattg tgacaaaata aacttattcc
     1861 gtggtgtctt tgcgtttctt ttatatgttg ccacctttat gtatgtattt tctacgtttg
     1921 ctaacatact gcgtaataag gagtcttaat catgccagtt cttttggcta gcgccgccct
     1981 ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg
     2041 aatggaagcc ggcggcacct cgctaacgga ttcaccactc caagaattgg agccaatcaa
     2101 ttcttgcgga gaactgtgaa tgcgcaaacc aacccttggc agaacatatc catcgcgtcc
     2161 gccatctcca gcagccgcac gcggcgcatc tcgggcagcg ttgggtcctg gccacgggtg
     2221 cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt
     2281 tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct
     2341 gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg
     2401 cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta
     2461 ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt
     2521 ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg
     2581 gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt
     2641 acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc
     2701 gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac
     2761 gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag
     2821 ctttaccgca ggatccgcga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     2881 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     2941 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3001 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3061 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3121 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3181 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3241 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcgcgg
     3301 atcctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3361 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3421 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     3481 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     3541 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc
     3601 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3661 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3721 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3781 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3841 acaggactat aaagatacca gggtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3901 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3961 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4021 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4081 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4141 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4201 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4261 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4321 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4381 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4441 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4501 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4561 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4621 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4681 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4741 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4801 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt
     4861 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4921 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4981 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5041 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5101 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg
     5161 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5221 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5281 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5341 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5401 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5461 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5521 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5581 cctttcgtct tcaagaattc ccgacaccat cgaatggtgc aaaacctttc gcggtatggc
     5641 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     5701 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     5761 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     5821 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     5881 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     5941 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     6001 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     6061 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     6121 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     6181 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     6241 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     6301 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     6361 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     6421 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     6481 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     6541 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     6601 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     6661 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     6721 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     6781 taagttagct cactcattag gcacaattct catgtttgac agcttatcat cgactgcacg
     6841 gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc
     6901 gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt
     6961 gcgccgacat cataacggtt ctggcaaata ttctgaaatg agctg
//
