LOCUS       sequence-191000-        6903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            NCK2-3/3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6903)
  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 6903)
  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_NCK2-3/3
FEATURES             Location/Qualifiers
     source          1..6903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     40..58
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        81..158
                     /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             159..1241
                     /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(178..197)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1484..1489,1490..1505,1506..1512)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1520..1536
                     /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     1525..1547
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1621..1638
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1654..2307
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2265..2287
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2314..2337
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2350..2367
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2581..2628
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4221..4676)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4358..4379)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4570..4589
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4718..4740)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4826..4966
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4881..4900
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4981..4998)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5152..5739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5910..6701,6702..6770))
                     /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     6533..6552
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6771..6875)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
       61 tttcgtcttc aagaattccc gacaccatcg aatggtgcaa aacctttcgc ggtatggcat
      121 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
      181 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
      241 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
      301 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
      361 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
      421 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
      481 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
      541 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
      601 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
      661 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
      721 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
      781 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
      841 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
      901 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
      961 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
     1021 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     1081 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
     1141 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     1201 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta
     1261 agttagctca ctcattaggc acaattctca tgtttgacag cttatcatcg actgcacggt
     1321 gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt
     1381 aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc
     1441 gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg
     1501 gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagccagtcc
     1561 gtttaggtgt tttcacgagc acttcaccaa caaggaccat agattatgaa aatcgaagaa
     1621 caccatcacc atcaccattc cagcggtaag cttatgtccc ctatactagg ttattggaaa
     1681 attaagggcc ttgtgcaacc cactcgactt cttttggaat atcttgaaga aaaatatgaa
     1741 gagcatttgt atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt
     1801 ttggagtttc ccaatcttcc ttattatatt gatggtgatg ttaaattaac acagtctatg
     1861 gccatcatac gttatatagc tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt
     1921 gcagagattt caatgcttga aggagcggtt ttggatatta gatacggtgt ttcgagaatt
     1981 gcatatagta aagactttga aactctcaaa gttgattttc ttagcaagct acctgaaatg
     2041 ctgaaaatgt tcgaagatcg tttatgtcat aaaacatatt taaatggtga tcatgtaacc
     2101 catcctgact tcatgttgta tgacgctctt gatgttgttt tatacatgga cccaatgtgc
     2161 ctggatgcgt tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat cccacaaatt
     2221 gataagtact tgaaatccag caagtatata gcatggcctt tgcagggctg gcaagccacg
     2281 tttggtggtg gcgaccatcc tccaaaatcg gatctggaag ttctgttcca ggggcccctg
     2341 tccagcggtc tggttccgcg tggttccggt accgcggccc agccggccca tgtggtgcag
     2401 accctgtatc cgtttagcag tgtgaccgaa gaagaactga attttgaaaa aggtgaaacg
     2461 atggaagtga ttgaaaaacc ggaaaacgat ccggaatggt ggaaatgcaa aaatgcgcgt
     2521 ggccaggtgg gtctggttcc gaaaaactat gtggttgtcc tgagcgcggc cgcataataa
     2581 accgatacaa ttaaaggctc cttttggagc cttttttttg gagattttca acgtgaaaaa
     2641 attattattc gcaattcctt tagttgttcc tttctattct cactccgctg aaactgttga
     2701 aagttgttta gcaaaacccc atacagaaaa ttcatttact aacgtctgga aagacgacaa
     2761 aactttagat cgttacgcta actatgaggg ctgtctgtgg aatgctacag gcgttgtagt
     2821 ttgtactggt gacgaaactc agtgtctagc tagagtggcg gtggctctgg ttccggtgat
     2881 tttgattatg aaaagatggc aaacgctaat aagggggcta tgaccgaaaa tgccgatgaa
     2941 aacgcgctac agtctgacgc taaaggcaaa cttgattctg tcgctactga ttacggtgct
     3001 gctatcgatg gtttcattgg tgacgtttcc ggccttgcta atggtaatgg tgctactggt
     3061 gattttgctg gctctaattc ccaaatggct caagtcggtg acggtgataa ttcaccttta
     3121 atgaataatt tccgtcaata tttaccttcc ctccctcaat cggttgaatg tcgccctttt
     3181 gtctttggcg ctggtaaacc atatgaattt tctattgatt gtgacaaaat aaacttattc
     3241 cgtggtgtct ttgcgtttct tttatatgtt gccaccttta tgtatgtatt ttctacgttt
     3301 gctaacatac tgcgtaataa ggagtcttaa tcatgccagt tcttttggct agcgccgccc
     3361 tataccttgt ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct
     3421 gaatggaagc cggcggcacc tcgctaacgg attcaccact ccaagaattg gagccaatca
     3481 attcttgcgg agaactgtga atgcgcaaac caacccttgg cagaacatat ccatcgcgtc
     3541 cgccatctcc agcagccgca cgcggcgcat ctcgggcagc gttgggtcct ggccacgggt
     3601 gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg
     3661 ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc
     3721 tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac
     3781 gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct
     3841 accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt
     3901 tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg
     3961 ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat
     4021 tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac
     4081 cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa
     4141 cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga
     4201 gctttaccgc aggatccgcg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     4261 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     4321 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     4381 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     4441 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     4501 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     4561 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     4621 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcgcg
     4681 gatcctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     4741 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     4801 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     4861 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     4921 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc
     4981 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5041 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5101 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5161 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5221 gacaggacta taaagatacc agggtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5281 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5341 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5401 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5461 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5521 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5581 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5641 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5701 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5761 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5821 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5881 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5941 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6001 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6061 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6121 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6181 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg
     6241 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6301 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6361 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6421 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6481 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc
     6541 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6601 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6661 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6721 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6781 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6841 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6901 gac
//
