LOCUS       sequence-215950-        6909 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Protein expression and purification of PLCG1_SH3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6909)
  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 6909)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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_PLCG1_SH3
FEATURES             Location/Qualifiers
     source          1..6909
                     /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      1104..1151
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2744..3199)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2881..2902)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3093..3112
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3241..3263)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3349..3489
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3404..3423
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3504..3521)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3675..4262)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4433..5224,5225..5293))
                     /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     5056..5075
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5294..5398)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5466..5484
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5507..5584
                     /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             5585..6667
                     /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(5604..5623)
                     /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 cggccacctt taaatgcgcg gtcaaagccc tgttcgatta taaagcacag cgtgaagacg
      961 aactgacgtt tattaaaagc gcgattatcc agaacgttga aaaacaagaa ggcggttggt
     1021 ggcgcggcga ttatggcggt aaaaaacaac tgtggttccc gtctaactac gtggaagaaa
     1081 tggttaatgc ggccgcataa taaaccgata caattaaagg ctccttttgg agcctttttt
     1141 ttggagattt tcaacgtgaa aaaattatta ttcgcaattc ctttagttgt tcctttctat
     1201 tctcactccg ctgaaactgt tgaaagttgt ttagcaaaac cccatacaga aaattcattt
     1261 actaacgtct ggaaagacga caaaacttta gatcgttacg ctaactatga gggctgtctg
     1321 tggaatgcta caggcgttgt agtttgtact ggtgacgaaa ctcagtgtct agctagagtg
     1381 gcggtggctc tggttccggt gattttgatt atgaaaagat ggcaaacgct aataaggggg
     1441 ctatgaccga aaatgccgat gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt
     1501 ctgtcgctac tgattacggt gctgctatcg atggtttcat tggtgacgtt tccggccttg
     1561 ctaatggtaa tggtgctact ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg
     1621 gtgacggtga taattcacct ttaatgaata atttccgtca atatttacct tccctccctc
     1681 aatcggttga atgtcgccct tttgtctttg gcgctggtaa accatatgaa ttttctattg
     1741 attgtgacaa aataaactta ttccgtggtg tctttgcgtt tcttttatat gttgccacct
     1801 ttatgtatgt attttctacg tttgctaaca tactgcgtaa taaggagtct taatcatgcc
     1861 agttcttttg gctagcgccg ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc
     1921 atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa cggattcacc
     1981 actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca aaccaaccct
     2041 tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg catctcgggc
     2101 agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta
     2161 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga
     2221 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc
     2281 cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc
     2341 tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc
     2401 tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta
     2461 ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc
     2521 ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca
     2581 tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca
     2641 ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa
     2701 tcgcttcacg accacgctga tgagctttac cgcaggatcc gcgaaattgt aagcgttaat
     2761 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     2821 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     2881 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     2941 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     3001 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     3061 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     3121 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     3181 gcgccgctac agggcgcgtc gcggatcctg cctcgcgcgt ttcggtgatg acggtgaaaa
     3241 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     3301 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     3361 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     3421 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     3481 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3541 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3601 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3661 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3721 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accagggttt ccccctggaa
     3781 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3841 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3901 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3961 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4021 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4081 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     4141 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4201 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4261 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4321 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4381 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     4441 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     4501 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     4561 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     4621 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     4681 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4741 ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4801 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4861 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4921 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4981 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5041 cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5101 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5161 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5221 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5281 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5341 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     5401 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     5461 ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attcccgaca ccatcgaatg
     5521 gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     5581 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     5641 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     5701 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     5761 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     5821 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     5881 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     5941 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     6001 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     6061 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     6121 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     6181 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     6241 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     6301 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     6361 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     6421 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     6481 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     6541 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     6601 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     6661 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcacaa ttctcatgtt
     6721 tgacagctta tcatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga
     6781 agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca
     6841 ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga
     6901 aatgagctg
//
