LOCUS       sequence-192539-        6894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            GRAP-2/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6894)
  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 6894)
  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_GRAP-2/2
FEATURES             Location/Qualifiers
     source          1..6894
                     /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      2548..2595
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4188..4643)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4325..4346)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4537..4556
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4685..4707)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4793..4933
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4848..4867
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4948..4965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5119..5706)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5877..6668,6669..6737))
                     /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     6500..6519
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6738..6842)
                     /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 ggcctgtttt gcacaggccc agtttgattt ctctgcccaa
     2401 gatccatctc aactgtcctt tcgtcgtggt gacattatcg aagttctgga acgtccagat
     2461 ccacattggt ggcgtggtcg ttcttgtggt cgtgttggtt tctttccgcg ttcctatgtt
     2521 cagccagttc atgcggccgc ataataaacc gatacaatta aaggctcctt ttggagcctt
     2581 ttttttggag attttcaacg tgaaaaaatt attattcgca attcctttag ttgttccttt
     2641 ctattctcac tccgctgaaa ctgttgaaag ttgtttagca aaaccccata cagaaaattc
     2701 atttactaac gtctggaaag acgacaaaac tttagatcgt tacgctaact atgagggctg
     2761 tctgtggaat gctacaggcg ttgtagtttg tactggtgac gaaactcagt gtctagctag
     2821 agtggcggtg gctctggttc cggtgatttt gattatgaaa agatggcaaa cgctaataag
     2881 ggggctatga ccgaaaatgc cgatgaaaac gcgctacagt ctgacgctaa aggcaaactt
     2941 gattctgtcg ctactgatta cggtgctgct atcgatggtt tcattggtga cgtttccggc
     3001 cttgctaatg gtaatggtgc tactggtgat tttgctggct ctaattccca aatggctcaa
     3061 gtcggtgacg gtgataattc acctttaatg aataatttcc gtcaatattt accttccctc
     3121 cctcaatcgg ttgaatgtcg cccttttgtc tttggcgctg gtaaaccata tgaattttct
     3181 attgattgtg acaaaataaa cttattccgt ggtgtctttg cgtttctttt atatgttgcc
     3241 acctttatgt atgtattttc tacgtttgct aacatactgc gtaataagga gtcttaatca
     3301 tgccagttct tttggctagc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg
     3361 gtgcatggag ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt
     3421 caccactcca agaattggag ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa
     3481 cccttggcag aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc
     3541 gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg
     3601 gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac
     3661 gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg
     3721 tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg
     3781 gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa
     3841 gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg
     3901 tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag
     3961 catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga
     4021 ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca
     4081 gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg
     4141 tgaatcgctt cacgaccacg ctgatgagct ttaccgcagg atccgcgaaa ttgtaagcgt
     4201 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     4261 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     4321 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     4381 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     4441 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     4501 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     4561 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     4621 taatgcgccg ctacagggcg cgtcgcggat cctgcctcgc gcgtttcggt gatgacggtg
     4681 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     4741 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     4801 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca
     4861 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     4921 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4981 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5041 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5101 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5161 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg gtttccccct
     5221 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5281 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5341 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5401 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5461 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5521 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     5581 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5641 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5701 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5761 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5821 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5881 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5941 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6001 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6061 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6121 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6181 gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6241 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6301 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6361 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6421 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6481 tgcccggcgt caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6541 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6601 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6661 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6721 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6781 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6841 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catg
//
