LOCUS       sequence-315910-        7009 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of SUB1_PC4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7009)
  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 7009)
  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_SUB1_PC4
FEATURES             Location/Qualifiers
     source          1..7009
                     /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      1204..1251
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2844..3299)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2981..3002)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3193..3212
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3341..3363)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3449..3589
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3504..3523
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3604..3621)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3775..4362)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4533..5324,5325..5393))
                     /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     5156..5175
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5394..5498)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5566..5584
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5607..5684
                     /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             5685..6767
                     /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(5704..5723)
                     /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 cggcctcccg tgatgacaac atgtttcaga ttggcaaaat gcgttatgtg agcgttcgcg
      961 atttcaaagg caaggtcctg attgatatcc gtgaatactg gatggacccg gaaggcgaaa
     1021 tgaaaccggg ccgcaagggt atcagcctga atccggaaca gtggtctcaa ctgaaagaac
     1081 aaattagtga tatcgatgac gcggttcgca agctggcggc cgcataataa accgatacaa
     1141 ttaatctaga tgcattcgcg aggtaccgcg gcccagccgg ccttttttgc ggccgcataa
     1201 taaaccgata caattaaagg ctccttttgg agcctttttt ttggagattt tcaacgtgaa
     1261 aaaattatta ttcgcaattc ctttagttgt tcctttctat tctcactccg ctgaaactgt
     1321 tgaaagttgt ttagcaaaac cccatacaga aaattcattt actaacgtct ggaaagacga
     1381 caaaacttta gatcgttacg ctaactatga gggctgtctg tggaatgcta caggcgttgt
     1441 agtttgtact ggtgacgaaa ctcagtgtct agctagagtg gcggtggctc tggttccggt
     1501 gattttgatt atgaaaagat ggcaaacgct aataaggggg ctatgaccga aaatgccgat
     1561 gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt
     1621 gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact
     1681 ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct
     1741 ttaatgaata atttccgtca atatttacct tccctccctc aatcggttga atgtcgccct
     1801 tttgtctttg gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta
     1861 ttccgtggtg tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttctacg
     1921 tttgctaaca tactgcgtaa taaggagtct taatcatgcc agttcttttg gctagcgccg
     1981 ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga
     2041 cctgaatgga agccggcggc acctcgctaa cggattcacc actccaagaa ttggagccaa
     2101 tcaattcttg cggagaactg tgaatgcgca aaccaaccct tggcagaaca tatccatcgc
     2161 gtccgccatc tccagcagcc gcacgcggcg catctcgggc agcgttgggt cctggccacg
     2221 ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac
     2281 tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac
     2341 gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga
     2401 aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg
     2461 gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat
     2521 ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa
     2581 ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat
     2641 cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa
     2701 aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact
     2761 caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga
     2821 tgagctttac cgcaggatcc gcgaaattgt aagcgttaat attttgttaa aattcgcgtt
     2881 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     2941 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     3001 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     3061 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     3121 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     3181 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     3241 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     3301 gcggatcctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     3361 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     3421 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     3481 gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     3541 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     3601 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     3661 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     3721 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3781 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3841 cccgacagga ctataaagat accagggttt ccccctggaa gctccctcgt gcgctctcct
     3901 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3961 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4021 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4081 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4141 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4201 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4261 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4321 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4381 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4441 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     4501 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     4561 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     4621 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     4681 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4741 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4801 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg
     4861 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4921 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4981 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5041 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5101 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat
     5161 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5221 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5281 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     5341 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     5401 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     5461 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     5521 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     5581 aggccctttc gtcttcaaga attcccgaca ccatcgaatg gtgcaaaacc tttcgcggta
     5641 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     5701 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     5761 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     5821 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     5881 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     5941 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     6001 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     6061 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     6121 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     6181 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     6241 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     6301 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     6361 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     6421 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     6481 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     6541 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     6601 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     6661 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     6721 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     6781 aatgtaagtt agctcactca ttaggcacaa ttctcatgtt tgacagctta tcatcgactg
     6841 cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta tggctgtgca
     6901 ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt
     6961 ttttgcgccg acatcataac ggttctggca aatattctga aatgagctg
//
