LOCUS       sequence-192609-        6921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            FRK-1/1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6921)
  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 6921)
  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_FRK-1/1
FEATURES             Location/Qualifiers
     source          1..6921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..22
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        45..122
                     /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             123..1205
                     /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(142..161)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1448..1453,1454..1469,1470..1476)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1484..1500
                     /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     1489..1511
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1585..1602
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1618..2271
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2229..2251
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2278..2301
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2314..2331
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2563..2610
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4203..4658)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4340..4361)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4552..4571
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4700..4722)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4808..4948
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4863..4882
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4963..4980)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5134..5721)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5892..6683,6684..6752))
                     /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     6515..6534
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6753..6857)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaaaataggc gtatcacgag gccctttcgt cttcaagaat tcccgacacc atcgaatggt
       61 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
      121 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
      181 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
      241 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
      301 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
      361 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
      421 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
      481 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
      541 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
      601 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
      661 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
      721 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
      781 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
      841 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
      901 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
      961 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     1021 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     1081 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     1141 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     1201 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacaatt ctcatgtttg
     1261 acagcttatc atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag
     1321 ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact
     1381 cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa
     1441 tgagctgttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag cggataacaa
     1501 tttcacacag gaaacagcca gtccgtttag gtgttttcac gagcacttca ccaacaagga
     1561 ccatagatta tgaaaatcga agaacaccat caccatcacc attccagcgg taagcttatg
     1621 tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg
     1681 gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg
     1741 cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt
     1801 gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg
     1861 ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat
     1921 attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat
     1981 tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca
     2041 tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt
     2101 gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa
     2161 cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg
     2221 cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa atcggatctg
     2281 gaagttctgt tccaggggcc cctgtccagc ggtctggttc cgcgtggttc cggtaccgcg
     2341 gcccagccgg cccattactt tgtcgccctg tttgattatc aagcacgtac cgccgaagat
     2401 ttgtcttttc gtgccggtga taaactgcaa gttctggata ccctgcatga aggctggtgg
     2461 tttgcacgtc atctggaaaa acgtcgtgat ggttcctctc aacagctgca aggttacatt
     2521 ccatccaact atgtcgcaga agatcgtgcg gccgcataat aaaccgatac aattaaaggc
     2581 tccttttgga gccttttttt tggagatttt caacgtgaaa aaattattat tcgcaattcc
     2641 tttagttgtt cctttctatt ctcactccgc tgaaactgtt gaaagttgtt tagcaaaacc
     2701 ccatacagaa aattcattta ctaacgtctg gaaagacgac aaaactttag atcgttacgc
     2761 taactatgag ggctgtctgt ggaatgctac aggcgttgta gtttgtactg gtgacgaaac
     2821 tcagtgtcta gctagagtgg cggtggctct ggttccggtg attttgatta tgaaaagatg
     2881 gcaaacgcta ataagggggc tatgaccgaa aatgccgatg aaaacgcgct acagtctgac
     2941 gctaaaggca aacttgattc tgtcgctact gattacggtg ctgctatcga tggtttcatt
     3001 ggtgacgttt ccggccttgc taatggtaat ggtgctactg gtgattttgc tggctctaat
     3061 tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt taatgaataa tttccgtcaa
     3121 tatttacctt ccctccctca atcggttgaa tgtcgccctt ttgtctttgg cgctggtaaa
     3181 ccatatgaat tttctattga ttgtgacaaa ataaacttat tccgtggtgt ctttgcgttt
     3241 cttttatatg ttgccacctt tatgtatgta ttttctacgt ttgctaacat actgcgtaat
     3301 aaggagtctt aatcatgcca gttcttttgg ctagcgccgc cctatacctt gtctgcctcc
     3361 ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa gccggcggca
     3421 cctcgctaac ggattcacca ctccaagaat tggagccaat caattcttgc ggagaactgt
     3481 gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct ccagcagccg
     3541 cacgcggcgc atctcgggca gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct
     3601 gtcgttgagg acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc
     3661 gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac
     3721 atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg
     3781 caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac
     3841 atctgtatta acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat
     3901 ccataccgcc agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt
     3961 aacccgtatc gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa
     4021 tcccccttac acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg
     4081 ctttatcaga agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga
     4141 acaggcagac atctgtgaat cgcttcacga ccacgctgat gagctttacc gcaggatccg
     4201 cgaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     4261 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     4321 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     4381 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     4441 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     4501 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     4561 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     4621 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg cggatcctgc ctcgcgcgtt
     4681 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     4741 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     4801 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta
     4861 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     4921 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct
     4981 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5041 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5101 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5161 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5221 ccagggtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5281 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5341 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5401 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5461 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5521 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     5581 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5641 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5701 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5761 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5821 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5881 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5941 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6001 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6061 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6121 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6181 agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt
     6241 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6301 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6361 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     6421 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     6481 cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact
     6541 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6601 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6661 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6721 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6781 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6841 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     6901 attatcatga cattaaccta t
//
