LOCUS       sequence-215954-        7482 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of PLAT_Trypsin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7482)
  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 7482)
  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_PLAT_Trypsin
FEATURES             Location/Qualifiers
     source          1..7482
                     /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      1677..1724
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3317..3772)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3454..3475)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3666..3685
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3814..3836)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3922..4062
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3977..3996
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4077..4094)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4248..4835)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5006..5797,5798..5866))
                     /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     5629..5648
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5867..5971)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6039..6057
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6080..6157
                     /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             6158..7240
                     /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(6177..6196)
                     /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 cggccattaa aggcggtctg tttgcggata tcgcctcaca tccgtggcag gcggccattt
      961 ttgcgaaaca ccgtcgctcg ccgggtgaac gtttcctgtg cggcggtatt ctgatcagct
     1021 cttgttggat tctgtctgca gctcattgct ttcaggaacg cttcccgccg catcacctga
     1081 ccgtgatcct gggtcgtacg tatcgcgtgg ttccgggcga agaagaacaa aaattcgaag
     1141 tggaaaaata cattgttcac aaagaattcg atgacgatac ctacgacaac gatatcgcgc
     1201 tgctgcagct gaaaagcgat agttcccgtt gtgcccaaga atcatcggtc gtgcgcaccg
     1261 tttgcctgcc gccggcagac ctgcagctgc cggattggac ggaatgtgaa ctgagtggct
     1321 atggtaaaca tgaagcactg agtccgttct actccgaacg tctgaaagaa gctcatgttc
     1381 gtctgtaccc gagctctcgc tgtacctccc aacacctgct gaaccgtacc gtcacggaca
     1441 atatgctgtg cgcaggtgat acgcgtagcg gcggtccgca ggcaaacctg cacgacgctt
     1501 gtcaaggcga ttctggcggt ccgctggtct gcctgaatga cggtcgcatg accctggtgg
     1561 gcattatcag ttggggcctg ggttgcggcc agaaagatgt tccgggcgtc tataccaaag
     1621 tgacgaacta cctggactgg atccgtgata atatgcgccc ggcggccgca taataaaccg
     1681 atacaattaa aggctccttt tggagccttt tttttggaga ttttcaacgt gaaaaaatta
     1741 ttattcgcaa ttcctttagt tgttcctttc tattctcact ccgctgaaac tgttgaaagt
     1801 tgtttagcaa aaccccatac agaaaattca tttactaacg tctggaaaga cgacaaaact
     1861 ttagatcgtt acgctaacta tgagggctgt ctgtggaatg ctacaggcgt tgtagtttgt
     1921 actggtgacg aaactcagtg tctagctaga gtggcggtgg ctctggttcc ggtgattttg
     1981 attatgaaaa gatggcaaac gctaataagg gggctatgac cgaaaatgcc gatgaaaacg
     2041 cgctacagtc tgacgctaaa ggcaaacttg attctgtcgc tactgattac ggtgctgcta
     2101 tcgatggttt cattggtgac gtttccggcc ttgctaatgg taatggtgct actggtgatt
     2161 ttgctggctc taattcccaa atggctcaag tcggtgacgg tgataattca cctttaatga
     2221 ataatttccg tcaatattta ccttccctcc ctcaatcggt tgaatgtcgc ccttttgtct
     2281 ttggcgctgg taaaccatat gaattttcta ttgattgtga caaaataaac ttattccgtg
     2341 gtgtctttgc gtttctttta tatgttgcca cctttatgta tgtattttct acgtttgcta
     2401 acatactgcg taataaggag tcttaatcat gccagttctt ttggctagcg ccgccctata
     2461 ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc cgggccacct cgacctgaat
     2521 ggaagccggc ggcacctcgc taacggattc accactccaa gaattggagc caatcaattc
     2581 ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga acatatccat cgcgtccgcc
     2641 atctccagca gccgcacgcg gcgcatctcg ggcagcgttg ggtcctggcc acgggtgcgc
     2701 atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag
     2761 cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg
     2821 acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg
     2881 aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc
     2941 tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc
     3001 tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca
     3061 tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc
     3121 cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc
     3181 cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag
     3241 ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt
     3301 taccgcagga tccgcgaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     3361 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     3421 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     3481 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     3541 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     3601 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     3661 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     3721 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcgcggatc
     3781 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     3841 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     3901 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     3961 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     4021 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg
     4081 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4141 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4201 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4261 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4321 ggactataaa gataccaggg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4381 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4441 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4501 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4561 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4621 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4681 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4741 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4801 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4861 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4921 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4981 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5041 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5101 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5161 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5221 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5281 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac
     5341 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5401 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5461 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5521 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5581 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc
     5641 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5701 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5761 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5821 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5881 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5941 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6001 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     6061 ttcgtcttca agaattcccg acaccatcga atggtgcaaa acctttcgcg gtatggcatg
     6121 atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga
     6181 tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag
     6241 ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat
     6301 tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac
     6361 ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga
     6421 tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa
     6481 agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct
     6541 ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct
     6601 tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg
     6661 actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc
     6721 attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa
     6781 tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca
     6841 aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca
     6901 gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat
     6961 ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac
     7021 catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc
     7081 tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac
     7141 caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     7201 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa
     7261 gttagctcac tcattaggca caattctcat gtttgacagc ttatcatcga ctgcacggtg
     7321 caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta
     7381 aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg
     7441 ccgacatcat aacggttctg gcaaatattc tgaaatgagc tg
//
