LOCUS       sequence-192213-        6897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            HCK-1/1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6897)
  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 6897)
  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_HCK-1/1
FEATURES             Location/Qualifiers
     source          1..6897
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..29
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        52..129
                     /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             130..1212
                     /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(149..168)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1455..1460,1461..1476,1477..1483)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1491..1507
                     /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     1496..1518
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1592..1609
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1625..2278
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2236..2258
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2285..2308
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2321..2338
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2546..2593
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4186..4641)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4323..4344)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4535..4554
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4683..4705)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4791..4931
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4846..4865
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4946..4963)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5117..5704)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5875..6666,6667..6735))
                     /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     6498..6517
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6736..6840)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattcc cgacaccatc
       61 gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
      121 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
      181 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
      241 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
      301 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
      361 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
      421 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
      481 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
      541 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
      601 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
      661 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
      721 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
      781 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
      841 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
      901 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
      961 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     1021 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     1081 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     1141 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     1201 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg cacaattctc
     1261 atgtttgaca gcttatcatc gactgcacgg tgcaccaatg cttctggcgt caggcagcca
     1321 tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt gtcgctcaag
     1381 gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat
     1441 tctgaaatga gctgttgaca attaatcatc ggctcgtata atgtgtggaa ttgtgagcgg
     1501 ataacaattt cacacaggaa acagccagtc cgtttaggtg ttttcacgag cacttcacca
     1561 acaaggacca tagattatga aaatcgaaga acaccatcac catcaccatt ccagcggtaa
     1621 gcttatgtcc cctatactag gttattggaa aattaagggc cttgtgcaac ccactcgact
     1681 tcttttggaa tatcttgaag aaaaatatga agagcatttg tatgagcgcg atgaaggtga
     1741 taaatggcga aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat
     1801 tgatggtgat gttaaattaa cacagtctat ggccatcata cgttatatag ctgacaagca
     1861 caacatgttg ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt
     1921 tttggatatt agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa
     1981 agttgatttt cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca
     2041 taaaacatat ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct
     2101 tgatgttgtt ttatacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt
     2161 taaaaaacgt attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat
     2221 agcatggcct ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaatc
     2281 ggatctggaa gttctgttcc aggggcccct gtccagcggt ctggttccgc gtggttccgg
     2341 taccgcggcc cagccggcca ttattgttgt ggcactgtat gactatgaag caatccatca
     2401 cgaagacctg tcgtttcaaa aaggcgatca aatggttgtt ctggaagaaa gcggcgaatg
     2461 gtggaaagcg cgttctctgg ccacccgcaa agaaggttat attccgagta actacgtggc
     2521 gcgtgttgat gcggccgcat aataaaccga tacaattaaa ggctcctttt ggagcctttt
     2581 ttttggagat tttcaacgtg aaaaaattat tattcgcaat tcctttagtt gttcctttct
     2641 attctcactc cgctgaaact gttgaaagtt gtttagcaaa accccataca gaaaattcat
     2701 ttactaacgt ctggaaagac gacaaaactt tagatcgtta cgctaactat gagggctgtc
     2761 tgtggaatgc tacaggcgtt gtagtttgta ctggtgacga aactcagtgt ctagctagag
     2821 tggcggtggc tctggttccg gtgattttga ttatgaaaag atggcaaacg ctaataaggg
     2881 ggctatgacc gaaaatgccg atgaaaacgc gctacagtct gacgctaaag gcaaacttga
     2941 ttctgtcgct actgattacg gtgctgctat cgatggtttc attggtgacg tttccggcct
     3001 tgctaatggt aatggtgcta ctggtgattt tgctggctct aattcccaaa tggctcaagt
     3061 cggtgacggt gataattcac ctttaatgaa taatttccgt caatatttac cttccctccc
     3121 tcaatcggtt gaatgtcgcc cttttgtctt tggcgctggt aaaccatatg aattttctat
     3181 tgattgtgac aaaataaact tattccgtgg tgtctttgcg tttcttttat atgttgccac
     3241 ctttatgtat gtattttcta cgtttgctaa catactgcgt aataaggagt cttaatcatg
     3301 ccagttcttt tggctagcgc cgccctatac cttgtctgcc tccccgcgtt gcgtcgcggt
     3361 gcatggagcc gggccacctc gacctgaatg gaagccggcg gcacctcgct aacggattca
     3421 ccactccaag aattggagcc aatcaattct tgcggagaac tgtgaatgcg caaaccaacc
     3481 cttggcagaa catatccatc gcgtccgcca tctccagcag ccgcacgcgg cgcatctcgg
     3541 gcagcgttgg gtcctggcca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc
     3601 taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt
     3661 gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt
     3721 tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga
     3781 tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc
     3841 gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt
     3901 taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca
     3961 tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg
     4021 catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga
     4081 cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg
     4141 aatcgcttca cgaccacgct gatgagcttt accgcaggat ccgcgaaatt gtaagcgtta
     4201 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     4261 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     4321 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     4381 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     4441 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     4501 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     4561 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     4621 atgcgccgct acagggcgcg tcgcggatcc tgcctcgcgc gtttcggtga tgacggtgaa
     4681 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     4741 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     4801 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     4861 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     4921 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4981 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5041 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5101 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5161 gctcaagtca gaggtggcga aacccgacag gactataaag ataccagggt ttccccctgg
     5221 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5281 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5341 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5401 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5461 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5521 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     5581 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5641 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5701 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5761 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5821 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5881 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5941 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6001 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6061 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6121 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6181 tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6241 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6301 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6361 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6421 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6481 cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6541 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6601 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6661 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6721 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6781 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6841 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacatt
//
