LOCUS       sequence-192599-        6894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            NCK2-1/3-SV1/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6894)
  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 6894)
  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_NCK2-1/3-SV1/2
FEATURES             Location/Qualifiers
     source          1..6894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        36..113
                     /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             114..1196
                     /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(133..152)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1439..1444,1445..1460,1461..1467)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1475..1491
                     /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     1480..1502
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1576..1593
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1609..2262
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2220..2242
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2269..2292
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2305..2322
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2527..2574
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4167..4622)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4304..4325)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4516..4535
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4664..4686)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4772..4912
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4827..4846
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4927..4944)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5098..5685)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5856..6647,6648..6716))
                     /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     6479..6498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6717..6821)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     join(6889..6894,1..13)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgtatcacga ggccctttcg tcttcaagaa ttcccgacac catcgaatgg tgcaaaacct
       61 ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac
      121 cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg
      181 tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg
      241 cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc
      301 tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga
      361 ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg
      421 gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga
      481 tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg
      541 ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt attttctccc
      601 atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg
      661 cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata
      721 aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca
      781 tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc
      841 tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc
      901 gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata
      961 tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc
     1021 gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac
     1081 tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg
     1141 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     1201 aacgcaatta atgtaagtta gctcactcat taggcacaat tctcatgttt gacagcttat
     1261 catcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat
     1321 ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg
     1381 gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt
     1441 gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca
     1501 ggaaacagcc agtccgttta ggtgttttca cgagcacttc accaacaagg accatagatt
     1561 atgaaaatcg aagaacacca tcaccatcac cattccagcg gtaagcttat gtcccctata
     1621 ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt ggaatatctt
     1681 gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa
     1741 aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg tgatgttaaa
     1801 ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat gttgggtggt
     1861 tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga tattagatac
     1921 ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga ttttcttagc
     1981 aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac atatttaaat
     2041 ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt tgttttatac
     2101 atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa acgtattgaa
     2161 gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg gcctttgcag
     2221 ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatct ggaagttctg
     2281 ttccaggggc ccctgtccag cggtctggtt ccgcgtggtt ccggtaccgc ggcccagccg
     2341 gccgtcatcg tcattgccaa atgggattac accgcacaac aagatcagga actggacatt
     2401 aaaaaaaacg aacgtctgtg gctgctggat gattctaaaa cctggtggcg tgttcgtaat
     2461 gcagcaaacc gtaccggtta tgttccatcc aactatgtcg aacgtaaaaa cgcggccgca
     2521 taataaaccg atacaattaa aggctccttt tggagccttt tttttggaga ttttcaacgt
     2581 gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc tattctcact ccgctgaaac
     2641 tgttgaaagt tgtttagcaa aaccccatac agaaaattca tttactaacg tctggaaaga
     2701 cgacaaaact ttagatcgtt acgctaacta tgagggctgt ctgtggaatg ctacaggcgt
     2761 tgtagtttgt actggtgacg aaactcagtg tctagctaga gtggcggtgg ctctggttcc
     2821 ggtgattttg attatgaaaa gatggcaaac gctaataagg gggctatgac cgaaaatgcc
     2881 gatgaaaacg cgctacagtc tgacgctaaa ggcaaacttg attctgtcgc tactgattac
     2941 ggtgctgcta tcgatggttt cattggtgac gtttccggcc ttgctaatgg taatggtgct
     3001 actggtgatt ttgctggctc taattcccaa atggctcaag tcggtgacgg tgataattca
     3061 cctttaatga ataatttccg tcaatattta ccttccctcc ctcaatcggt tgaatgtcgc
     3121 ccttttgtct ttggcgctgg taaaccatat gaattttcta ttgattgtga caaaataaac
     3181 ttattccgtg gtgtctttgc gtttctttta tatgttgcca cctttatgta tgtattttct
     3241 acgtttgcta acatactgcg taataaggag tcttaatcat gccagttctt ttggctagcg
     3301 ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc cgggccacct
     3361 cgacctgaat ggaagccggc ggcacctcgc taacggattc accactccaa gaattggagc
     3421 caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga acatatccat
     3481 cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg ggcagcgttg ggtcctggcc
     3541 acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct
     3601 tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa
     3661 aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct
     3721 ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg
     3781 ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt
     3841 gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag
     3901 taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg
     3961 tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga
     4021 aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa
     4081 actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc
     4141 tgatgagctt taccgcagga tccgcgaaat tgtaagcgtt aatattttgt taaaattcgc
     4201 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     4261 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     4321 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     4381 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     4441 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     4501 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     4561 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     4621 gtcgcggatc ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     4681 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     4741 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     4801 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     4861 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     4921 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4981 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5041 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5101 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5161 aaacccgaca ggactataaa gataccaggg tttccccctg gaagctccct cgtgcgctct
     5221 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5281 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5341 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5401 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5461 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5521 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5581 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5641 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5701 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5761 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5821 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5881 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5941 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6001 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6061 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6121 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc
     6181 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6241 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6301 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6361 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6421 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat
     6481 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6541 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6601 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6661 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6721 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6781 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6841 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa tagg
//
