LOCUS       sequence-192358-        6912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            NCK1-1/3.
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_NCK1-1/3
FEATURES             Location/Qualifiers
     source          1..6912
                     /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      2566..2613
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4206..4661)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4343..4364)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4555..4574
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4703..4725)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4811..4951
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4866..4885
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4966..4983)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5137..5724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5895..6686,6687..6755))
                     /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     6518..6537
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6756..6860)
                     /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 ggccatggcc cagggcgcgc tggttgtcgt tgtcgccaaa
     2401 tttgattatg tcgcccagca agaacaggaa ctggacatta aaaagaacga acgtctgtgg
     2461 ctgctggatg attctaaatc ctggtggcgt gttcgtaact ccatgaacaa aaccggtttc
     2521 gtcccatcca actatgtcga acgtaaaaac gcggccgcat aataaaccga tacaattaaa
     2581 ggctcctttt ggagcctttt ttttggagat tttcaacgtg aaaaaattat tattcgcaat
     2641 tcctttagtt gttcctttct attctcactc cgctgaaact gttgaaagtt gtttagcaaa
     2701 accccataca gaaaattcat ttactaacgt ctggaaagac gacaaaactt tagatcgtta
     2761 cgctaactat gagggctgtc tgtggaatgc tacaggcgtt gtagtttgta ctggtgacga
     2821 aactcagtgt ctagctagag tggcggtggc tctggttccg gtgattttga ttatgaaaag
     2881 atggcaaacg ctaataaggg ggctatgacc gaaaatgccg atgaaaacgc gctacagtct
     2941 gacgctaaag gcaaacttga ttctgtcgct actgattacg gtgctgctat cgatggtttc
     3001 attggtgacg tttccggcct tgctaatggt aatggtgcta ctggtgattt tgctggctct
     3061 aattcccaaa tggctcaagt cggtgacggt gataattcac ctttaatgaa taatttccgt
     3121 caatatttac cttccctccc tcaatcggtt gaatgtcgcc cttttgtctt tggcgctggt
     3181 aaaccatatg aattttctat tgattgtgac aaaataaact tattccgtgg tgtctttgcg
     3241 tttcttttat atgttgccac ctttatgtat gtattttcta cgtttgctaa catactgcgt
     3301 aataaggagt cttaatcatg ccagttcttt tggctagcgc cgccctatac cttgtctgcc
     3361 tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg gaagccggcg
     3421 gcacctcgct aacggattca ccactccaag aattggagcc aatcaattct tgcggagaac
     3481 tgtgaatgcg caaaccaacc cttggcagaa catatccatc gcgtccgcca tctccagcag
     3541 ccgcacgcgg cgcatctcgg gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct
     3601 cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc agaatgaatc
     3661 accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac
     3721 aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc
     3781 ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc
     3841 tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg
     3901 catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc
     3961 agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag
     4021 aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc ttaacatggc
     4081 ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc tggacgcgga
     4141 tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt accgcaggat
     4201 ccgcgaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     4261 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     4321 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     4381 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     4441 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     4501 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     4561 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     4621 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcgcggatcc tgcctcgcgc
     4681 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     4741 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     4801 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     4861 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     4921 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc
     4981 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5041 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5101 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5161 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5221 ataccagggt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5281 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5341 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5401 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5461 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5521 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     5581 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5641 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5701 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5761 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5821 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5881 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5941 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6001 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6061 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6121 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6181 aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt
     6241 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccata
     6301 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6361 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6421 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6481 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga
     6541 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6601 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6661 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6721 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6781 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6841 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     6901 attattatca tg
//
