LOCUS       sequence-215956-        6903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of APLP2_Kunitz-BPTI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6903)
  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 6903)
  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_APLP2_Kunitz-BPTI
FEATURES             Location/Qualifiers
     source          1..6903
                     /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      1098..1145
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2738..3193)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2875..2896)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3087..3106
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3235..3257)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3343..3483
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3398..3417
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3498..3515)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3669..4256)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4427..5218,5219..5287))
                     /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     5050..5069
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5288..5392)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5460..5478
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5501..5578
                     /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             5579..6661
                     /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(5598..5617)
                     /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 cggccgtgaa agcagtttgc agccaggaag ctatgaccgg tccgtgtcgt gcagtcatgc
      961 cgcgctggta ttttgatctg tctaagggta aatgcgtgcg tttcatttac ggcggttgtg
     1021 gcggtaaccg caacaatttt gaaagtgaag actattgcat ggcggtttgt aaagccatga
     1081 tcgcggccgc ataataaacc gatacaatta aaggctcctt ttggagcctt ttttttggag
     1141 attttcaacg tgaaaaaatt attattcgca attcctttag ttgttccttt ctattctcac
     1201 tccgctgaaa ctgttgaaag ttgtttagca aaaccccata cagaaaattc atttactaac
     1261 gtctggaaag acgacaaaac tttagatcgt tacgctaact atgagggctg tctgtggaat
     1321 gctacaggcg ttgtagtttg tactggtgac gaaactcagt gtctagctag agtggcggtg
     1381 gctctggttc cggtgatttt gattatgaaa agatggcaaa cgctaataag ggggctatga
     1441 ccgaaaatgc cgatgaaaac gcgctacagt ctgacgctaa aggcaaactt gattctgtcg
     1501 ctactgatta cggtgctgct atcgatggtt tcattggtga cgtttccggc cttgctaatg
     1561 gtaatggtgc tactggtgat tttgctggct ctaattccca aatggctcaa gtcggtgacg
     1621 gtgataattc acctttaatg aataatttcc gtcaatattt accttccctc cctcaatcgg
     1681 ttgaatgtcg cccttttgtc tttggcgctg gtaaaccata tgaattttct attgattgtg
     1741 acaaaataaa cttattccgt ggtgtctttg cgtttctttt atatgttgcc acctttatgt
     1801 atgtattttc tacgtttgct aacatactgc gtaataagga gtcttaatca tgccagttct
     1861 tttggctagc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag
     1921 ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt caccactcca
     1981 agaattggag ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa cccttggcag
     2041 aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc gggcagcgtt
     2101 gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg
     2161 cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac
     2221 tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt
     2281 tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc
     2341 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat
     2401 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca
     2461 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct
     2521 cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg
     2581 accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg
     2641 cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt
     2701 cacgaccacg ctgatgagct ttaccgcagg atccgcgaaa ttgtaagcgt taatattttg
     2761 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     2821 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     2881 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     2941 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     3001 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     3061 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     3121 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     3181 ctacagggcg cgtcgcggat cctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     3241 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     3301 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     3361 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     3421 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     3481 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3541 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3601 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3661 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3721 cagaggtggc gaaacccgac aggactataa agataccagg gtttccccct ggaagctccc
     3781 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3841 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3901 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3961 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4021 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4081 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     4141 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4201 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4261 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4321 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4381 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4441 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4501 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4561 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4621 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4681 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4741 ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4801 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4861 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4921 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4981 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5041 caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5101 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5161 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5221 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5281 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5341 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5401 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     5461 ataggcgtat cacgaggccc tttcgtcttc aagaattccc gacaccatcg aatggtgcaa
     5521 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     5581 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     5641 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     5701 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     5761 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     5821 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     5881 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     5941 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     6001 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     6061 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     6121 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     6181 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     6241 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     6301 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     6361 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
     6421 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     6481 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
     6541 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
     6601 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     6661 agcgcaacgc aattaatgta agttagctca ctcattaggc acaattctca tgtttgacag
     6721 cttatcatcg actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt
     6781 ggtatggctg tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg
     6841 ttctggataa tgttttttgc gccgacatca taacggttct ggcaaatatt ctgaaatgag
     6901 ctg
//
