LOCUS       sequence-192226-        6912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            NCK2-2/3.
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_NCK2-2/3
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..22
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        45..122
                     /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             123..1205
                     /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(142..161)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1448..1453,1454..1469,1470..1476)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1484..1500
                     /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     1489..1511
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1585..1602
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1618..2271
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2229..2251
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2278..2301
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2314..2331
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2554..2601
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4194..4649)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4331..4352)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4543..4562
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4691..4713)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4799..4939
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4854..4873
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4954..4971)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5125..5712)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5883..6674,6675..6743))
                     /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     6506..6525
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6744..6848)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaaataggc gtatcacgag gccctttcgt cttcaagaat tcccgacacc atcgaatggt
       61 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
      121 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
      181 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
      241 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
      301 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
      361 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
      421 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
      481 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
      541 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
      601 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
      661 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
      721 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
      781 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
      841 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
      901 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
      961 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     1021 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     1081 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     1141 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     1201 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacaatt ctcatgtttg
     1261 acagcttatc atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag
     1321 ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact
     1381 cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa
     1441 tgagctgttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag cggataacaa
     1501 tttcacacag gaaacagcca gtccgtttag gtgttttcac gagcacttca ccaacaagga
     1561 ccatagatta tgaaaatcga agaacaccat caccatcacc attccagcgg taagcttatg
     1621 tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg
     1681 gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg
     1741 cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt
     1801 gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg
     1861 ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat
     1921 attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat
     1981 tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca
     2041 tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt
     2101 gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa
     2161 cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg
     2221 cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa atcggatctg
     2281 gaagttctgt tccaggggcc cctgtccagc ggtctggttc cgcgtggttc cggtaccgcg
     2341 gcccagccgg ccatggccca gggcgcgctg attccggcct ttgtcaaatt tgcctacgtc
     2401 gccgaacgtg aagatgaact gtccctggtt aaaggttctc gtgtcaccgt catggaaaaa
     2461 tgttccgatg gttggtggcg tggttcttac aatggtcaga ttggttggtt tccgtccaac
     2521 tacgttctgg aagaagtcgc ggccgcataa taaaccgata caattaaagg ctccttttgg
     2581 agcctttttt ttggagattt tcaacgtgaa aaaattatta ttcgcaattc ctttagttgt
     2641 tcctttctat tctcactccg ctgaaactgt tgaaagttgt ttagcaaaac cccatacaga
     2701 aaattcattt actaacgtct ggaaagacga caaaacttta gatcgttacg ctaactatga
     2761 gggctgtctg tggaatgcta caggcgttgt agtttgtact ggtgacgaaa ctcagtgtct
     2821 agctagagtg gcggtggctc tggttccggt gattttgatt atgaaaagat ggcaaacgct
     2881 aataaggggg ctatgaccga aaatgccgat gaaaacgcgc tacagtctga cgctaaaggc
     2941 aaacttgatt ctgtcgctac tgattacggt gctgctatcg atggtttcat tggtgacgtt
     3001 tccggccttg ctaatggtaa tggtgctact ggtgattttg ctggctctaa ttcccaaatg
     3061 gctcaagtcg gtgacggtga taattcacct ttaatgaata atttccgtca atatttacct
     3121 tccctccctc aatcggttga atgtcgccct tttgtctttg gcgctggtaa accatatgaa
     3181 ttttctattg attgtgacaa aataaactta ttccgtggtg tctttgcgtt tcttttatat
     3241 gttgccacct ttatgtatgt attttctacg tttgctaaca tactgcgtaa taaggagtct
     3301 taatcatgcc agttcttttg gctagcgccg ccctatacct tgtctgcctc cccgcgttgc
     3361 gtcgcggtgc atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa
     3421 cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca
     3481 aaccaaccct tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg
     3541 catctcgggc agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     3601 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     3661 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     3721 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     3781 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     3841 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     3901 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     3961 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     4021 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     4081 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     4141 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcaggatcc gcgaaattgt
     4201 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     4261 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     4321 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     4381 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     4441 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     4501 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     4561 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     4621 cgcgcttaat gcgccgctac agggcgcgtc gcggatcctg cctcgcgcgt ttcggtgatg
     4681 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4741 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     4801 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     4861 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     4921 gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4981 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5041 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5101 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5161 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accagggttt
     5221 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5281 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5341 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5401 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5461 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5521 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     5581 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5641 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5701 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5761 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5821 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5881 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5941 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6001 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6061 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6121 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6181 aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6241 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatatt gtgcaaaaaa
     6301 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6361 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6421 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6481 tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg
     6541 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6601 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6661 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6721 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6781 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6841 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     6901 acattaacct at
//
