LOCUS       sequence-192372-        6915 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            GRB2-1/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6915)
  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 6915)
  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_GRB2-1/2
FEATURES             Location/Qualifiers
     source          1..6915
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..34
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        57..134
                     /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             135..1217
                     /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(154..173)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1460..1465,1466..1481,1482..1488)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1496..1512
                     /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     1501..1523
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1597..1614
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1630..2283
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2241..2263
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2290..2313
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2326..2343
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2569..2616
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4209..4664)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4346..4367)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4558..4577
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4706..4728)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4814..4954
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4869..4888
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4969..4986)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5140..5727)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5898..6689,6690..6758))
                     /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     6521..6540
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6759..6863)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attcccgaca
       61 ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
      121 tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct
      181 cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg
      241 aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac
      301 tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc
      361 cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
      421 tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg
      481 cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg
      541 tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca
      601 tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg
      661 cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
      721 tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac
      781 gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg
      841 gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg
      901 ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata
      961 ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc
     1021 tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca
     1081 atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa
     1141 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     1201 tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcacaa
     1261 ttctcatgtt tgacagctta tcatcgactg cacggtgcac caatgcttct ggcgtcaggc
     1321 agccatcgga agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc
     1381 tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca
     1441 aatattctga aatgagctgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg
     1501 agcggataac aatttcacac aggaaacagc cagtccgttt aggtgttttc acgagcactt
     1561 caccaacaag gaccatagat tatgaaaatc gaagaacacc atcaccatca ccattccagc
     1621 ggtaagctta tgtcccctat actaggttat tggaaaatta agggccttgt gcaacccact
     1681 cgacttcttt tggaatatct tgaagaaaaa tatgaagagc atttgtatga gcgcgatgaa
     1741 ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa tcttccttat
     1801 tatattgatg gtgatgttaa attaacacag tctatggcca tcatacgtta tatagctgac
     1861 aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat gcttgaagga
     1921 gcggttttgg atattagata cggtgtttcg agaattgcat atagtaaaga ctttgaaact
     1981 ctcaaagttg attttcttag caagctacct gaaatgctga aaatgttcga agatcgttta
     2041 tgtcataaaa catatttaaa tggtgatcat gtaacccatc ctgacttcat gttgtatgac
     2101 gctcttgatg ttgttttata catggaccca atgtgcctgg atgcgttccc aaaattagtt
     2161 tgttttaaaa aacgtattga agctatccca caaattgata agtacttgaa atccagcaag
     2221 tatatagcat ggcctttgca gggctggcaa gccacgtttg gtggtggcga ccatcctcca
     2281 aaatcggatc tggaagttct gttccagggg cccctgtcca gcggtctggt tccgcgtggt
     2341 tccggtaccg cggcccagcc ggccatggcc cagggcgcgc tgatggaagc cattgccaaa
     2401 tatgatttca aagccaccgc cgatgatgaa ctgtctttca aacgtggtga tattctgaaa
     2461 gtcctgaacg aagaatgcga tcagaactgg tacaaagccg aactgaacgg taaagatggt
     2521 ttcattccga aaaactacat tgaaatgaaa ccggcggccg cataataaac cgatacaatt
     2581 aaaggctcct tttggagcct tttttttgga gattttcaac gtgaaaaaat tattattcgc
     2641 aattccttta gttgttcctt tctattctca ctccgctgaa actgttgaaa gttgtttagc
     2701 aaaaccccat acagaaaatt catttactaa cgtctggaaa gacgacaaaa ctttagatcg
     2761 ttacgctaac tatgagggct gtctgtggaa tgctacaggc gttgtagttt gtactggtga
     2821 cgaaactcag tgtctagcta gagtggcggt ggctctggtt ccggtgattt tgattatgaa
     2881 aagatggcaa acgctaataa gggggctatg accgaaaatg ccgatgaaaa cgcgctacag
     2941 tctgacgcta aaggcaaact tgattctgtc gctactgatt acggtgctgc tatcgatggt
     3001 ttcattggtg acgtttccgg ccttgctaat ggtaatggtg ctactggtga ttttgctggc
     3061 tctaattccc aaatggctca agtcggtgac ggtgataatt cacctttaat gaataatttc
     3121 cgtcaatatt taccttccct ccctcaatcg gttgaatgtc gcccttttgt ctttggcgct
     3181 ggtaaaccat atgaattttc tattgattgt gacaaaataa acttattccg tggtgtcttt
     3241 gcgtttcttt tatatgttgc cacctttatg tatgtatttt ctacgtttgc taacatactg
     3301 cgtaataagg agtcttaatc atgccagttc ttttggctag cgccgcccta taccttgtct
     3361 gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga atggaagccg
     3421 gcggcacctc gctaacggat tcaccactcc aagaattgga gccaatcaat tcttgcggag
     3481 aactgtgaat gcgcaaacca acccttggca gaacatatcc atcgcgtccg ccatctccag
     3541 cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt
     3601 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga
     3661 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc
     3721 aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
     3781 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac
     3841 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg
     3901 ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc
     3961 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa
     4021 cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat
     4081 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
     4141 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
     4201 gatccgcgaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     4261 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     4321 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     4381 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     4441 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     4501 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     4561 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     4621 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcgcgga tcctgcctcg
     4681 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     4741 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     4801 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     4861 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     4921 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
     4981 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5041 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5101 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5161 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5221 aagataccag ggtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5281 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5341 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5401 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5461 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5521 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     5581 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5641 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5701 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5761 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5821 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5881 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5941 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6001 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6061 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6121 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6181 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
     6241 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6301 atattgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6361 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6421 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6481 atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc
     6541 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6601 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6661 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6721 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6781 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6841 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     6901 accattatta tcatg
//
