LOCUS       sequence-192272-        6912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            GRB2-2/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6912)
  AUTHORS   Teyra J, Huang H, Jain S, Guan X, Dong A, Liu Y, Tempel W, Min J, 
            Tong Y, Kim PM, Bader GD, Sidhu SS
  TITLE     Comprehensive Analysis of the Human SH3 Domain Family Reveals a Wide
            Variety of Non-canonical Specificities.
  JOURNAL   Structure. 2017 Oct 3;25(10):1598-1610.e3. doi: 
            10.1016/j.str.2017.07.017. Epub 2017 Sep 7.
  PUBMED    28890361
REFERENCE   2  (bases 1 to 6912)
  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"; doi: 
            "10.1016/j.str.2017.07.017"; journalName: "Structure"; date: 
            "2017-10-3- 3"; volume: "25"; issue: "10"; pages: "1598-1610.e3"
COMMENT     Sequence Label: pHH0103_GRB2-2/2
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..32
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        55..132
                     /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             133..1215
                     /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(152..171)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1458..1463,1464..1479,1480..1486)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1494..1510
                     /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     1499..1521
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1595..1612
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1628..2281
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2239..2261
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2288..2311
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2324..2341
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2564..2611
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4204..4659)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4341..4362)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4553..4572
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4701..4723)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4809..4949
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4864..4883
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4964..4981)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5135..5722)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5893..6684,6685..6753))
                     /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     6516..6535
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6754..6858)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat tcccgacacc
       61 atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
      121 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
      181 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
      241 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
      301 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
      361 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
      421 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
      481 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
      541 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
      601 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
      661 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
      721 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
      781 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
      841 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
      901 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
      961 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     1021 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     1081 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     1141 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     1201 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacaatt
     1261 ctcatgtttg acagcttatc atcgactgca cggtgcacca atgcttctgg cgtcaggcag
     1321 ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc
     1381 aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa
     1441 tattctgaaa tgagctgttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag
     1501 cggataacaa tttcacacag gaaacagcca gtccgtttag gtgttttcac gagcacttca
     1561 ccaacaagga ccatagatta tgaaaatcga agaacaccat caccatcacc attccagcgg
     1621 taagcttatg tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg
     1681 acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg
     1741 tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta
     1801 tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa
     1861 gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc
     1921 ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct
     1981 caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg
     2041 tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc
     2101 tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg
     2161 ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta
     2221 tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa
     2281 atcggatctg gaagttctgt tccaggggcc cctgtccagc ggtctggttc cgcgtggttc
     2341 cggtaccgcg gcccagccgg ccatggccca gggcgcgctg acctatgttc aggccctgtt
     2401 tgattttgat ccgcaggaag atggtgaact gggttttcgt cgtggtgatt tcattcatgt
     2461 catggataac tccgatccga attggtggaa aggtgcatgt catggtcaga ccggtatgtt
     2521 tccacgtaac tatgttaccc cggtcaacgc ggccgcataa taaaccgata caattaaagg
     2581 ctccttttgg agcctttttt ttggagattt tcaacgtgaa aaaattatta ttcgcaattc
     2641 ctttagttgt tcctttctat tctcactccg ctgaaactgt tgaaagttgt ttagcaaaac
     2701 cccatacaga aaattcattt actaacgtct ggaaagacga caaaacttta gatcgttacg
     2761 ctaactatga gggctgtctg tggaatgcta caggcgttgt agtttgtact ggtgacgaaa
     2821 ctcagtgtct agctagagtg gcggtggctc tggttccggt gattttgatt atgaaaagat
     2881 ggcaaacgct aataaggggg ctatgaccga aaatgccgat gaaaacgcgc tacagtctga
     2941 cgctaaaggc aaacttgatt ctgtcgctac tgattacggt gctgctatcg atggtttcat
     3001 tggtgacgtt tccggccttg ctaatggtaa tggtgctact ggtgattttg ctggctctaa
     3061 ttcccaaatg gctcaagtcg gtgacggtga taattcacct ttaatgaata atttccgtca
     3121 atatttacct tccctccctc aatcggttga atgtcgccct tttgtctttg gcgctggtaa
     3181 accatatgaa ttttctattg attgtgacaa aataaactta ttccgtggtg tctttgcgtt
     3241 tcttttatat gttgccacct ttatgtatgt attttctacg tttgctaaca tactgcgtaa
     3301 taaggagtct taatcatgcc agttcttttg gctagcgccg ccctatacct tgtctgcctc
     3361 cccgcgttgc gtcgcggtgc atggagccgg gccacctcga cctgaatgga agccggcggc
     3421 acctcgctaa cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg
     3481 tgaatgcgca aaccaaccct tggcagaaca tatccatcgc gtccgccatc tccagcagcc
     3541 gcacgcggcg catctcgggc agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc
     3601 tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac
     3661 cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa
     3721 catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct
     3781 gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta
     3841 catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca
     3901 tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag
     3961 taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa
     4021 atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc
     4081 gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg
     4141 aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac cgcaggatcc
     4201 gcgaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     4261 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     4321 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     4381 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     4441 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     4501 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     4561 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     4621 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcggatcctg cctcgcgcgt
     4681 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     4741 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     4801 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact
     4861 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     4921 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     4981 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5041 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5101 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5161 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5221 accagggttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5281 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5341 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5401 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5461 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5521 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     5581 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5641 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5701 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5761 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5821 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5881 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5941 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6001 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6061 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6121 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6181 tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg
     6241 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatatt
     6301 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6361 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6421 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6481 gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac
     6541 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6601 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6661 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6721 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6781 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6841 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     6901 tattatcatg ac
//
