LOCUS       sequence-213681-        7113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of FRS2_IRS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7113)
  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 7113)
  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_FRS2_IRS
FEATURES             Location/Qualifiers
     source          1..7113
                     /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      1308..1355
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2948..3403)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3085..3106)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3297..3316
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3445..3467)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3553..3693
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3608..3627
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3708..3725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3879..4466)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4637..5428,5429..5497))
                     /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     5260..5279
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5498..5602)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5670..5688
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5711..5788
                     /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             5789..6871
                     /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(5808..5827)
                     /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 cggccccgga taaggacact gttccagata atcaccgtaa caaattcaaa gttattaacg
      961 tcgacgacga tggcaacgaa ctgggctctg gtattatgga actgacggac actgaactga
     1021 tcctgtacac ccgcaaacgt gattctgtaa aatggcatta cctgtgcctg cgtcgctatg
     1081 gctacgacag caatctgttt tccttcgaat ctggtcgccg ttgccagacc ggtcagggta
     1141 tcttcgcttt taaatgtgca cgtgcggaag agctgttcaa catgctgcag gaaatcatgc
     1201 aaaacaactc catcaacgtt gttgaagagc cggtggtgga acgtaacaac caccagaccg
     1261 aactggaggt accgcgtacc ccgcgcaccc ctgcggccgc ataataaacc gatacaatta
     1321 aaggctcctt ttggagcctt ttttttggag attttcaacg tgaaaaaatt attattcgca
     1381 attcctttag ttgttccttt ctattctcac tccgctgaaa ctgttgaaag ttgtttagca
     1441 aaaccccata cagaaaattc atttactaac gtctggaaag acgacaaaac tttagatcgt
     1501 tacgctaact atgagggctg tctgtggaat gctacaggcg ttgtagtttg tactggtgac
     1561 gaaactcagt gtctagctag agtggcggtg gctctggttc cggtgatttt gattatgaaa
     1621 agatggcaaa cgctaataag ggggctatga ccgaaaatgc cgatgaaaac gcgctacagt
     1681 ctgacgctaa aggcaaactt gattctgtcg ctactgatta cggtgctgct atcgatggtt
     1741 tcattggtga cgtttccggc cttgctaatg gtaatggtgc tactggtgat tttgctggct
     1801 ctaattccca aatggctcaa gtcggtgacg gtgataattc acctttaatg aataatttcc
     1861 gtcaatattt accttccctc cctcaatcgg ttgaatgtcg cccttttgtc tttggcgctg
     1921 gtaaaccata tgaattttct attgattgtg acaaaataaa cttattccgt ggtgtctttg
     1981 cgtttctttt atatgttgcc acctttatgt atgtattttc tacgtttgct aacatactgc
     2041 gtaataagga gtcttaatca tgccagttct tttggctagc gccgccctat accttgtctg
     2101 cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg
     2161 cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt cttgcggaga
     2221 actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc catctccagc
     2281 agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg
     2341 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     2401 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca
     2461 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg
     2521 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     2581 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
     2641 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
     2701 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
     2761 agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg
     2821 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg
     2881 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagg
     2941 atccgcgaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     3001 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     3061 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     3121 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     3181 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     3241 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     3301 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     3361 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcgcggat cctgcctcgc
     3421 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     3481 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     3541 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     3601 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     3661 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     3721 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     3781 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     3841 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     3901 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     3961 agataccagg gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4021 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4081 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4141 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4201 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4261 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     4321 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4381 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4441 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4501 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4561 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4621 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4681 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4741 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4801 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4861 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4921 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     4981 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5041 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5101 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5161 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5221 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     5281 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5341 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5401 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5461 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5521 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5581 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     5641 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     5701 aagaattccc gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc
     5761 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     5821 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     5881 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     5941 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     6001 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     6061 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     6121 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     6181 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     6241 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     6301 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     6361 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     6421 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     6481 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     6541 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     6601 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     6661 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     6721 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     6781 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     6841 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     6901 ctcattaggc acaattctca tgtttgacag cttatcatcg actgcacggt gcaccaatgc
     6961 ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc
     7021 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca
     7081 taacggttct ggcaaatatt ctgaaatgag ctg
//
