LOCUS       sequence-214930-        7059 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of IRS1_IRS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7059)
  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 7059)
  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_IRS1_IRS
FEATURES             Location/Qualifiers
     source          1..7059
                     /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      1254..1301
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2894..3349)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3031..3052)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3243..3262
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3391..3413)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3499..3639
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3554..3573
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3654..3671)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3825..4412)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4583..5374,5375..5443))
                     /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     5206..5225
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5444..5548)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5616..5634
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5657..5734
                     /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             5735..6817
                     /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(5754..5773)
                     /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 cggccggtcc ggcatttaag gaagtgtggc aagttattct gaaaccgaag ggtctgggtc
      961 aaacgaaaaa cctgattggc atttaccgtc tgtgcctgac cagcaaaacg atttctttcg
     1021 tgaagctgaa ctctgaagcg gcggcagtgg ttctgcagct gatgaacatt cgtcgctgtg
     1081 gccatagtga aaactttttc tttatcgaag ttggtcgttc cgcagtcacc ggtccgggtg
     1141 aattctggat gcaggttgat gactcagtcg tggcgcaaaa tatgcacgaa acgatcctgg
     1201 aagctatgcg cgcgatgtcg gatgaatttc gtccgcgcgc ggccgcataa taaaccgata
     1261 caattaaagg ctccttttgg agcctttttt ttggagattt tcaacgtgaa aaaattatta
     1321 ttcgcaattc ctttagttgt tcctttctat tctcactccg ctgaaactgt tgaaagttgt
     1381 ttagcaaaac cccatacaga aaattcattt actaacgtct ggaaagacga caaaacttta
     1441 gatcgttacg ctaactatga gggctgtctg tggaatgcta caggcgttgt agtttgtact
     1501 ggtgacgaaa ctcagtgtct agctagagtg gcggtggctc tggttccggt gattttgatt
     1561 atgaaaagat ggcaaacgct aataaggggg ctatgaccga aaatgccgat gaaaacgcgc
     1621 tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt gctgctatcg
     1681 atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact ggtgattttg
     1741 ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct ttaatgaata
     1801 atttccgtca atatttacct tccctccctc aatcggttga atgtcgccct tttgtctttg
     1861 gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta ttccgtggtg
     1921 tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttctacg tttgctaaca
     1981 tactgcgtaa taaggagtct taatcatgcc agttcttttg gctagcgccg ccctatacct
     2041 tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga cctgaatgga
     2101 agccggcggc acctcgctaa cggattcacc actccaagaa ttggagccaa tcaattcttg
     2161 cggagaactg tgaatgcgca aaccaaccct tggcagaaca tatccatcgc gtccgccatc
     2221 tccagcagcc gcacgcggcg catctcgggc agcgttgggt cctggccacg ggtgcgcatg
     2281 atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag
     2341 aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc
     2401 tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag
     2461 tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt
     2521 ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg
     2581 tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt
     2641 tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc
     2701 atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt
     2761 aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg
     2821 gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac
     2881 cgcaggatcc gcgaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     2941 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     3001 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     3061 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     3121 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     3181 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     3241 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     3301 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcggatcctg
     3361 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     3421 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     3481 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     3541 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     3601 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     3661 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3721 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3781 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3841 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3901 ctataaagat accagggttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3961 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4021 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4081 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4141 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4201 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4261 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4321 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4381 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4441 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4501 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4561 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4621 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4681 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4741 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4801 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4861 cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct
     4921 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4981 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5041 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5101 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5161 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac
     5221 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5281 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5341 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5401 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5461 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5521 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     5581 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     5641 gtcttcaaga attcccgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata
     5701 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     5761 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     5821 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     5881 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     5941 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     6001 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     6061 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     6121 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     6181 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     6241 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     6301 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     6361 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     6421 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     6481 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     6541 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     6601 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     6661 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     6721 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6781 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     6841 agctcactca ttaggcacaa ttctcatgtt tgacagctta tcatcgactg cacggtgcac
     6901 caatgcttct ggcgtcaggc agccatcgga agctgtggta tggctgtgca ggtcgtaaat
     6961 cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg
     7021 acatcataac ggttctggca aatattctga aatgagctg
//
