LOCUS       sequence-215927-        6900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of GRB2_SH3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6900)
  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 6900)
  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_GRB2_SH3
FEATURES             Location/Qualifiers
     source          1..6900
                     /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      1095..1142
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(2735..3190)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2872..2893)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3084..3103
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3232..3254)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3340..3480
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3395..3414
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3495..3512)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3666..4253)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(4424..5215,5216..5284))
                     /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     5047..5066
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5285..5389)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5457..5475
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5498..5575
                     /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             5576..6658
                     /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(5595..5614)
                     /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 cggccatgga agcgattgcc aaatatgatt ttaaagcaac cgctgatgac gaactgagct
      961 tcaaacgtgg cgatatcctg aaagtgctga acgaagaatg cgaccagaat tggtataaag
     1021 ccgaactgaa cggcaaagac ggtttcatcc cgaaaaacta catcgaaatg aaaccgcatg
     1081 cggccgcata ataaaccgat acaattaaag gctccttttg gagccttttt tttggagatt
     1141 ttcaacgtga aaaaattatt attcgcaatt cctttagttg ttcctttcta ttctcactcc
     1201 gctgaaactg ttgaaagttg tttagcaaaa ccccatacag aaaattcatt tactaacgtc
     1261 tggaaagacg acaaaacttt agatcgttac gctaactatg agggctgtct gtggaatgct
     1321 acaggcgttg tagtttgtac tggtgacgaa actcagtgtc tagctagagt ggcggtggct
     1381 ctggttccgg tgattttgat tatgaaaaga tggcaaacgc taataagggg gctatgaccg
     1441 aaaatgccga tgaaaacgcg ctacagtctg acgctaaagg caaacttgat tctgtcgcta
     1501 ctgattacgg tgctgctatc gatggtttca ttggtgacgt ttccggcctt gctaatggta
     1561 atggtgctac tggtgatttt gctggctcta attcccaaat ggctcaagtc ggtgacggtg
     1621 ataattcacc tttaatgaat aatttccgtc aatatttacc ttccctccct caatcggttg
     1681 aatgtcgccc ttttgtcttt ggcgctggta aaccatatga attttctatt gattgtgaca
     1741 aaataaactt attccgtggt gtctttgcgt ttcttttata tgttgccacc tttatgtatg
     1801 tattttctac gtttgctaac atactgcgta ataaggagtc ttaatcatgc cagttctttt
     1861 ggctagcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg
     1921 ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga
     1981 attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac
     2041 atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg
     2101 tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg
     2161 ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc
     2221 tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg
     2281 taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca
     2341 ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga
     2401 ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa
     2461 cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt
     2521 ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc
     2581 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt
     2641 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac
     2701 gaccacgctg atgagcttta ccgcaggatc cgcgaaattg taagcgttaa tattttgtta
     2761 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     2821 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     2881 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     2941 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     3001 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     3061 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     3121 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     3181 cagggcgcgt cgcggatcct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     3241 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     3301 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     3361 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     3421 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     3481 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3541 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3601 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3661 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3721 aggtggcgaa acccgacagg actataaaga taccagggtt tccccctgga agctccctcg
     3781 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3841 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3901 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3961 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4021 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4081 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     4141 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4201 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4261 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4321 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     4381 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     4441 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     4501 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     4561 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     4621 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4681 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg
     4741 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4801 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4861 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4921 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4981 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5041 cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5101 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5161 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5221 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5281 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     5341 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     5401 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     5461 ggcgtatcac gaggcccttt cgtcttcaag aattcccgac accatcgaat ggtgcaaaac
     5521 ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa
     5581 accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg
     5641 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat
     5701 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt
     5761 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc
     5821 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag
     5881 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct
     5941 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa
     6001 tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc
     6061 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat
     6121 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca
     6181 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc
     6241 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat
     6301 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct
     6361 gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta
     6421 tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga
     6481 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc
     6541 actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt
     6601 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     6661 gcaacgcaat taatgtaagt tagctcactc attaggcaca attctcatgt ttgacagctt
     6721 atcatcgact gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt
     6781 atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc
     6841 tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg
//
