LOCUS       sequence-191845-        6918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            CRK-2/2-SV2/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6918)
  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 6918)
  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-SV2/2
FEATURES             Location/Qualifiers
     source          1..6918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..36
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        59..136
                     /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             137..1219
                     /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(156..175)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1462..1467,1468..1483,1484..1490)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1498..1514
                     /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     1503..1525
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1599..1616
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1632..2285
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2243..2265
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2292..2315
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2328..2345
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2574..2621
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4214..4669)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4351..4372)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4563..4582
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4711..4733)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4819..4959
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4874..4893
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4974..4991)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5145..5732)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5903..6694,6695..6763))
                     /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     6526..6545
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6764..6868)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaattcccga
       61 caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
      121 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
      181 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
      241 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
      301 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
      361 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
      421 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
      481 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
      541 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
      601 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
      661 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
      721 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
      781 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
      841 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
      901 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
      961 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     1021 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     1081 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
     1141 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     1201 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     1261 aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag
     1321 gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc
     1381 gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg
     1441 caaatattct gaaatgagct gttgacaatt aatcatcggc tcgtataatg tgtggaattg
     1501 tgagcggata acaatttcac acaggaaaca gccagtccgt ttaggtgttt tcacgagcac
     1561 ttcaccaaca aggaccatag attatgaaaa tcgaagaaca ccatcaccat caccattcca
     1621 gcggtaagct tatgtcccct atactaggtt attggaaaat taagggcctt gtgcaaccca
     1681 ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg
     1741 aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt
     1801 attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg
     1861 acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag
     1921 gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa
     1981 ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt
     2041 tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg
     2101 acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag
     2161 tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca
     2221 agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc
     2281 caaaatcgga tctggaagtt ctgttccagg ggcccctgtc cagcggtctg gttccgcgtg
     2341 gttccggtac cgcggcccag ccggccatgg cccagggcgc gctgatctac gcccgtgtca
     2401 ttcaaaaacg tgtcccgaac gcctatgaca aaaccgcact ggcactggaa gttggtgaac
     2461 tggttaaagt taccaaaatc aacgtttccg gtcaatggga aggcggctgc aatggtaaac
     2521 gtggtcattt tccgtttacc catgtccgtc tgctggatgc ggccgcataa taaaccgata
     2581 caattaaagg ctccttttgg agcctttttt ttggagattt tcaacgtgaa aaaattatta
     2641 ttcgcaattc ctttagttgt tcctttctat tctcactccg ctgaaactgt tgaaagttgt
     2701 ttagcaaaac cccatacaga aaattcattt actaacgtct ggaaagacga caaaacttta
     2761 gatcgttacg ctaactatga gggctgtctg tggaatgcta caggcgttgt agtttgtact
     2821 ggtgacgaaa ctcagtgtct agctagagtg gcggtggctc tggttccggt gattttgatt
     2881 atgaaaagat ggcaaacgct aataaggggg ctatgaccga aaatgccgat gaaaacgcgc
     2941 tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt gctgctatcg
     3001 atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact ggtgattttg
     3061 ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct ttaatgaata
     3121 atttccgtca atatttacct tccctccctc aatcggttga atgtcgccct tttgtctttg
     3181 gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta ttccgtggtg
     3241 tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttctacg tttgctaaca
     3301 tactgcgtaa taaggagtct taatcatgcc agttcttttg gctagcgccg ccctatacct
     3361 tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga cctgaatgga
     3421 agccggcggc acctcgctaa cggattcacc actccaagaa ttggagccaa tcaattcttg
     3481 cggagaactg tgaatgcgca aaccaaccct tggcagaaca tatccatcgc gtccgccatc
     3541 tccagcagcc gcacgcggcg catctcgggc agcgttgggt cctggccacg ggtgcgcatg
     3601 atcgtgctcc tgtcgttgag gacccggcta ggctggcggg gttgccttac tggttagcag
     3661 aatgaatcac cgatacgcga gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc
     3721 tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag
     3781 tcagcgccct gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt
     3841 ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat ttttctctgg
     3901 tcccgccgca tccataccgc cagttgttta ccctcacaac gttccagtaa ccgggcatgt
     3961 tcatcatcag taacccgtat cgtgagcatc ctctctcgtt tcatcggtat cattaccccc
     4021 atgaacagaa atccccctta cacggaggca tcagtgacca aacaggaaaa aaccgccctt
     4081 aacatggccc gctttatcag aagccagaca ttaacgcttc tggagaaact caacgagctg
     4141 gacgcggatg aacaggcaga catctgtgaa tcgcttcacg accacgctga tgagctttac
     4201 cgcaggatcc gcgaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     4261 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     4321 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     4381 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     4441 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     4501 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     4561 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     4621 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcggatcctg
     4681 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     4741 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     4801 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     4861 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     4921 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     4981 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5041 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5101 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5161 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5221 ctataaagat accagggttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     5281 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     5341 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     5401 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     5461 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     5521 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     5581 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     5641 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     5701 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     5761 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5821 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5881 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5941 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     6001 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     6061 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     6121 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     6181 cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct
     6241 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     6301 cccccatatt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     6361 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     6421 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     6481 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac
     6541 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     6601 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     6661 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6721 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6781 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     6841 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     6901 aagaaaccat tattatca
//
