LOCUS       sequence-215957-        7629 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Protein expression and purification of ACR_Trypsin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7629)
  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 7629)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_ACR_Trypsin
FEATURES             Location/Qualifiers
     source          1..7629
                     /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      1824..1871
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3464..3919)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3601..3622)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3813..3832
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3961..3983)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4069..4209
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4124..4143
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4224..4241)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4395..4982)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5153..5944,5945..6013))
                     /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     5776..5795
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6014..6118)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6186..6204
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6227..6304
                     /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             6305..7387
                     /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(6324..6343)
                     /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 cggccatcgt gggcggtaaa gcagcacagc atggtgcatg gccgtggatg gttagcctgc
      961 aaatttttac ctataactct catcgttacc acacgtgcgg cggtagcctg ctgaattctc
     1021 gctgggtcct gaccgcagct cattgttttg tgggtaaaaa caatgttcac gattggcgtc
     1081 tggtgttcgg cgctaaagaa atcacgtatg gtaacaataa accggttaaa gcgccgctgc
     1141 aggaacgcta tgtcgaaaaa atcatcatcc atgaaaaata caacagtgcg accgaaggca
     1201 atgacatcgc cctggttgaa atcacgccgc cgatttcctg cggtcgtttt attggcccgg
     1261 gttgtctgcc gcacttcaaa gcaggtctgc cgcgtggtag tcaatcctgc tgggttgccg
     1321 gctggggtta tatcgaagaa aaagcaccgc gtccgagctc tatcctgatg gaagctcgcg
     1381 tcgatctgat tgatctggac ctgtgcaaca gtacccagtg gtataatggc cgtgtccaac
     1441 cgacgaacgt gtgtgcaggc tacccggttg gtaaaattga tacctgccag ggtgactctg
     1501 gcggtccgct gatgtgtaaa gattcaaaag aatcggccta cgtggttgtc ggcatcacca
     1561 gctggggcgt gggttgtgca cgtgctaaac gcccgggtat ttataccgca acgtggccgt
     1621 acctgaactg gatcgcaagt aaaattggct ccaatgctct gcgcatgatt cagagcgcga
     1681 ccccgccgcc gccgaccacg cgtccgccgc cgatccgccc gccgttttca catccgattt
     1741 cggcccacct gccgtggtat tttcagccgc cgccgcgtcc gctgccgccg cgtccgccgg
     1801 cggcccaagc ggccgcataa taaaccgata caattaaagg ctccttttgg agcctttttt
     1861 ttggagattt tcaacgtgaa aaaattatta ttcgcaattc ctttagttgt tcctttctat
     1921 tctcactccg ctgaaactgt tgaaagttgt ttagcaaaac cccatacaga aaattcattt
     1981 actaacgtct ggaaagacga caaaacttta gatcgttacg ctaactatga gggctgtctg
     2041 tggaatgcta caggcgttgt agtttgtact ggtgacgaaa ctcagtgtct agctagagtg
     2101 gcggtggctc tggttccggt gattttgatt atgaaaagat ggcaaacgct aataaggggg
     2161 ctatgaccga aaatgccgat gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt
     2221 ctgtcgctac tgattacggt gctgctatcg atggtttcat tggtgacgtt tccggccttg
     2281 ctaatggtaa tggtgctact ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg
     2341 gtgacggtga taattcacct ttaatgaata atttccgtca atatttacct tccctccctc
     2401 aatcggttga atgtcgccct tttgtctttg gcgctggtaa accatatgaa ttttctattg
     2461 attgtgacaa aataaactta ttccgtggtg tctttgcgtt tcttttatat gttgccacct
     2521 ttatgtatgt attttctacg tttgctaaca tactgcgtaa taaggagtct taatcatgcc
     2581 agttcttttg gctagcgccg ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc
     2641 atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa cggattcacc
     2701 actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca aaccaaccct
     2761 tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg catctcgggc
     2821 agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta
     2881 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga
     2941 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc
     3001 cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc
     3061 tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc
     3121 tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta
     3181 ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc
     3241 ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca
     3301 tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca
     3361 ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa
     3421 tcgcttcacg accacgctga tgagctttac cgcaggatcc gcgaaattgt aagcgttaat
     3481 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     3541 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     3601 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     3661 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     3721 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     3781 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     3841 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     3901 gcgccgctac agggcgcgtc gcggatcctg cctcgcgcgt ttcggtgatg acggtgaaaa
     3961 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     4021 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     4081 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     4141 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     4201 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4261 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4321 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4381 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4441 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accagggttt ccccctggaa
     4501 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4561 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4621 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4681 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4741 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4801 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     4861 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4921 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4981 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5041 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5101 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5161 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5221 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5281 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5341 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5401 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5461 ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5521 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5581 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5641 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5701 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5761 cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5821 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5881 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5941 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6001 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6061 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6121 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     6181 ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attcccgaca ccatcgaatg
     6241 gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     6301 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     6361 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     6421 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     6481 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     6541 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     6601 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     6661 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     6721 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     6781 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     6841 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     6901 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     6961 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     7021 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     7081 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     7141 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     7201 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     7261 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     7321 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     7381 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcacaa ttctcatgtt
     7441 tgacagctta tcatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga
     7501 agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca
     7561 ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga
     7621 aatgagctg
//
