LOCUS       sequence-192282-        6912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            NCK1-2/3.
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_NCK1-2/3
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..40
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        63..140
                     /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             141..1223
                     /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(160..179)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1466..1471,1472..1487,1488..1494)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1502..1518
                     /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     1507..1529
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1603..1620
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1636..2289
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2247..2269
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2296..2319
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2332..2349
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2572..2619
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4212..4667)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4349..4370)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4561..4580
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4709..4731)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4817..4957
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4872..4891
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4972..4989)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5143..5730)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5901..6692,6693..6761))
                     /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     6524..6543
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6762..6866)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattc
       61 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
      121 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      181 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      241 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      301 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      361 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      421 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      481 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      541 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      601 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      661 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      721 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      781 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      841 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      901 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
      961 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
     1021 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1081 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1141 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1201 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1261 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1321 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1381 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1441 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1501 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1561 gcacttcacc aacaaggacc atagattatg aaaatcgaag aacaccatca ccatcaccat
     1621 tccagcggta agcttatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
     1681 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
     1741 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
     1801 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
     1861 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
     1921 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
     1981 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
     2041 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
     2101 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
     2161 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
     2221 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
     2281 cctccaaaat cggatctgga agttctgttc caggggcccc tgtccagcgg tctggttccg
     2341 cgtggttccg gtaccgcggc ccagccggcc atggcccagg gcgcgctgat gccggcctac
     2401 gtcaaattta actacatggc cgaacgtgaa gatgaactgt ccctgatcaa aggtacgaaa
     2461 gttattgtta tggaaaaatg ttccgatggt tggtggcgtg gttcttacaa tggtcaagtc
     2521 ggttggtttc catccaacta tgttaccgaa gaaggtgcgg ccgcataata aaccgataca
     2581 attaaaggct ccttttggag cctttttttt ggagattttc aacgtgaaaa aattattatt
     2641 cgcaattcct ttagttgttc ctttctattc tcactccgct gaaactgttg aaagttgttt
     2701 agcaaaaccc catacagaaa attcatttac taacgtctgg aaagacgaca aaactttaga
     2761 tcgttacgct aactatgagg gctgtctgtg gaatgctaca ggcgttgtag tttgtactgg
     2821 tgacgaaact cagtgtctag ctagagtggc ggtggctctg gttccggtga ttttgattat
     2881 gaaaagatgg caaacgctaa taagggggct atgaccgaaa atgccgatga aaacgcgcta
     2941 cagtctgacg ctaaaggcaa acttgattct gtcgctactg attacggtgc tgctatcgat
     3001 ggtttcattg gtgacgtttc cggccttgct aatggtaatg gtgctactgg tgattttgct
     3061 ggctctaatt cccaaatggc tcaagtcggt gacggtgata attcaccttt aatgaataat
     3121 ttccgtcaat atttaccttc cctccctcaa tcggttgaat gtcgcccttt tgtctttggc
     3181 gctggtaaac catatgaatt ttctattgat tgtgacaaaa taaacttatt ccgtggtgtc
     3241 tttgcgtttc ttttatatgt tgccaccttt atgtatgtat tttctacgtt tgctaacata
     3301 ctgcgtaata aggagtctta atcatgccag ttcttttggc tagcgccgcc ctataccttg
     3361 tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc tgaatggaag
     3421 ccggcggcac ctcgctaacg gattcaccac tccaagaatt ggagccaatc aattcttgcg
     3481 gagaactgtg aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt ccgccatctc
     3541 cagcagccgc acgcggcgca tctcgggcag cgttgggtcc tggccacggg tgcgcatgat
     3601 cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa
     3661 tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg
     3721 agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc
     3781 agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg
     3841 aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc
     3901 ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc
     3961 atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat
     4021 gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa
     4081 catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga
     4141 cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg
     4201 caggatccgc gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     4261 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     4321 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     4381 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     4441 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     4501 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     4561 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4621 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcgc ggatcctgcc
     4681 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     4741 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     4801 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     4861 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     4921 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     4981 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5041 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5101 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5161 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5221 ataaagatac cagggtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5281 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5341 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5401 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5461 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5521 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5581 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5641 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5701 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5761 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5821 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5881 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5941 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6001 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6061 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6121 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6181 ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg
     6241 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6301 cccatattgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6361 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6421 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6481 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat
     6541 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6601 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6661 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6721 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6781 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6841 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     6901 gaaaccatta tt
//
