LOCUS       sequence-191786-        6993 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            CRK-2/2-SV1/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6993)
  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 6993)
  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_CRK-2/2-SV1/2
FEATURES             Location/Qualifiers
     source          1..6993
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..43
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        66..143
                     /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             144..1226
                     /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(163..182)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1469..1474,1475..1490,1491..1497)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1505..1521
                     /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     1510..1532
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1606..1623
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1639..2292
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2250..2272
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2299..2322
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2335..2352
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2656..2703
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4296..4751)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4433..4454)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4645..4664
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4793..4815)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4901..5041
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4956..4975
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5056..5073)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5227..5814)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5985..6776,6777..6845))
                     /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     6608..6627
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6846..6950)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcaagaa
       61 ttcccgacac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
      121 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
      181 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
      241 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
      301 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
      361 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
      421 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
      481 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
      541 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
      601 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
      661 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
      721 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
      781 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
      841 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
      901 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
      961 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     1021 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     1081 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     1141 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     1201 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     1261 taggcacaat tctcatgttt gacagcttat catcgactgc acggtgcacc aatgcttctg
     1321 gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat
     1381 tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg
     1441 gttctggcaa atattctgaa atgagctgtt gacaattaat catcggctcg tataatgtgt
     1501 ggaattgtga gcggataaca atttcacaca ggaaacagcc agtccgttta ggtgttttca
     1561 cgagcacttc accaacaagg accatagatt atgaaaatcg aagaacacca tcaccatcac
     1621 cattccagcg gtaagcttat gtcccctata ctaggttatt ggaaaattaa gggccttgtg
     1681 caacccactc gacttctttt ggaatatctt gaagaaaaat atgaagagca tttgtatgag
     1741 cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat
     1801 cttccttatt atattgatgg tgatgttaaa ttaacacagt ctatggccat catacgttat
     1861 atagctgaca agcacaacat gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg
     1921 cttgaaggag cggttttgga tattagatac ggtgtttcga gaattgcata tagtaaagac
     1981 tttgaaactc tcaaagttga ttttcttagc aagctacctg aaatgctgaa aatgttcgaa
     2041 gatcgtttat gtcataaaac atatttaaat ggtgatcatg taacccatcc tgacttcatg
     2101 ttgtatgacg ctcttgatgt tgttttatac atggacccaa tgtgcctgga tgcgttccca
     2161 aaattagttt gttttaaaaa acgtattgaa gctatcccac aaattgataa gtacttgaaa
     2221 tccagcaagt atatagcatg gcctttgcag ggctggcaag ccacgtttgg tggtggcgac
     2281 catcctccaa aatcggatct ggaagttctg ttccaggggc ccctgtccag cggtctggtt
     2341 ccgcgtggtt ccggtaccgc ggcccagccg gccggtggtc cggaaccggg tccgtatgcc
     2401 caaccgtcag tgaatacccc gctgccgaat ctgcaaaatg gtccgatcta tgctcgtgtc
     2461 attcagaaac gtgttccgaa cgcgtatgat aaaaccgcac tggcactgga agtcggtgaa
     2521 ctggtgaaag ttacgaaaat caacgtgagc ggccaatggg agggtgaatg caatggcaaa
     2581 cgtggtcatt ttccgttcac ccacgtccgc ctgctggacc agcaaaatcc ggatgaagac
     2641 gcggccgcat aataaaccga tacaattaaa ggctcctttt ggagcctttt ttttggagat
     2701 tttcaacgtg aaaaaattat tattcgcaat tcctttagtt gttcctttct attctcactc
     2761 cgctgaaact gttgaaagtt gtttagcaaa accccataca gaaaattcat ttactaacgt
     2821 ctggaaagac gacaaaactt tagatcgtta cgctaactat gagggctgtc tgtggaatgc
     2881 tacaggcgtt gtagtttgta ctggtgacga aactcagtgt ctagctagag tggcggtggc
     2941 tctggttccg gtgattttga ttatgaaaag atggcaaacg ctaataaggg ggctatgacc
     3001 gaaaatgccg atgaaaacgc gctacagtct gacgctaaag gcaaacttga ttctgtcgct
     3061 actgattacg gtgctgctat cgatggtttc attggtgacg tttccggcct tgctaatggt
     3121 aatggtgcta ctggtgattt tgctggctct aattcccaaa tggctcaagt cggtgacggt
     3181 gataattcac ctttaatgaa taatttccgt caatatttac cttccctccc tcaatcggtt
     3241 gaatgtcgcc cttttgtctt tggcgctggt aaaccatatg aattttctat tgattgtgac
     3301 aaaataaact tattccgtgg tgtctttgcg tttcttttat atgttgccac ctttatgtat
     3361 gtattttcta cgtttgctaa catactgcgt aataaggagt cttaatcatg ccagttcttt
     3421 tggctagcgc cgccctatac cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc
     3481 gggccacctc gacctgaatg gaagccggcg gcacctcgct aacggattca ccactccaag
     3541 aattggagcc aatcaattct tgcggagaac tgtgaatgcg caaaccaacc cttggcagaa
     3601 catatccatc gcgtccgcca tctccagcag ccgcacgcgg cgcatctcgg gcagcgttgg
     3661 gtcctggcca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg
     3721 gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg
     3781 ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc
     3841 gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
     3901 aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg
     3961 accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca
     4021 acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg
     4081 tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac
     4141 caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct
     4201 tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca
     4261 cgaccacgct gatgagcttt accgcaggat ccgcgaaatt gtaagcgtta atattttgtt
     4321 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     4381 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     4441 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     4501 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     4561 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     4621 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     4681 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     4741 acagggcgcg tcgcggatcc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac
     4801 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     4861 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac
     4921 gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag
     4981 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     5041 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5101 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5161 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5221 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5281 gaggtggcga aacccgacag gactataaag ataccagggt ttccccctgg aagctccctc
     5341 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5401 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5461 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5521 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5581 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5641 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     5701 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5761 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5821 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5881 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5941 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6001 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6061 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6121 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6181 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6241 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6301 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6361 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6421 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6481 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6541 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6601 acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6661 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6721 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6781 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6841 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6901 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6961 cgaaaagtgc cacctgacgt ctaagaaacc att
//
