LOCUS       sequence-192228-        6918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            CRKL-2/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6918)
  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 6918)
  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_CRKL-2/2
FEATURES             Location/Qualifiers
     source          1..6918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     12..30
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        53..130
                     /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             131..1213
                     /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(150..169)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1456..1461,1462..1477,1478..1484)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1492..1508
                     /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     1497..1519
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1593..1610
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1626..2279
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2237..2259
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2286..2309
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2322..2339
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2568..2615
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4208..4663)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4345..4366)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4557..4576
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4705..4727)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4813..4953
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4868..4887
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4968..4985)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5139..5726)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5897..6688,6689..6757))
                     /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     6520..6539
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6758..6862)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattc ccgacaccat
       61 cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
      121 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
      181 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
      241 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
      301 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
      361 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
      421 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
      481 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
      541 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
      601 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
      661 gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg
      721 tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga
      781 aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat
      841 cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat
      901 taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga
      961 agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg
     1021 gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca
     1081 gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc
     1141 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     1201 aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag gcacaattct
     1261 catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc
     1321 atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa
     1381 ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata
     1441 ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga attgtgagcg
     1501 gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga gcacttcacc
     1561 aacaaggacc atagattatg aaaatcgaag aacaccatca ccatcaccat tccagcggta
     1621 agcttatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac
     1681 ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg
     1741 ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata
     1801 ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc
     1861 acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg
     1921 ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca
     1981 aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc
     2041 ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc
     2101 ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt
     2161 ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata
     2221 tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaat
     2281 cggatctgga agttctgttc caggggcccc tgtccagcgg tctggttccg cgtggttccg
     2341 gtaccgcggc ccagccggcc atggcccagg gcgcgctggt tttcgccaaa gccattcaga
     2401 aacgtgttcc gtgcgcctat gacaaaaccg cactggcact ggaagttggt gatattgtta
     2461 aagtcacccg tatgaacatt aacggtcaat gggaaggtga agtcaacggt cgtaaaggtc
     2521 tctttccgtt cacccatgtc aaaatcttcg atgcggccgc ataataaacc gatacaatta
     2581 aaggctcctt ttggagcctt ttttttggag attttcaacg tgaaaaaatt attattcgca
     2641 attcctttag ttgttccttt ctattctcac tccgctgaaa ctgttgaaag ttgtttagca
     2701 aaaccccata cagaaaattc atttactaac gtctggaaag acgacaaaac tttagatcgt
     2761 tacgctaact atgagggctg tctgtggaat gctacaggcg ttgtagtttg tactggtgac
     2821 gaaactcagt gtctagctag agtggcggtg gctctggttc cggtgatttt gattatgaaa
     2881 agatggcaaa cgctaataag ggggctatga ccgaaaatgc cgatgaaaac gcgctacagt
     2941 ctgacgctaa aggcaaactt gattctgtcg ctactgatta cggtgctgct atcgatggtt
     3001 tcattggtga cgtttccggc cttgctaatg gtaatggtgc tactggtgat tttgctggct
     3061 ctaattccca aatggctcaa gtcggtgacg gtgataattc acctttaatg aataatttcc
     3121 gtcaatattt accttccctc cctcaatcgg ttgaatgtcg cccttttgtc tttggcgctg
     3181 gtaaaccata tgaattttct attgattgtg acaaaataaa cttattccgt ggtgtctttg
     3241 cgtttctttt atatgttgcc acctttatgt atgtattttc tacgtttgct aacatactgc
     3301 gtaataagga gtcttaatca tgccagttct tttggctagc gccgccctat accttgtctg
     3361 cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg
     3421 cggcacctcg ctaacggatt caccactcca agaattggag ccaatcaatt cttgcggaga
     3481 actgtgaatg cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc catctccagc
     3541 agccgcacgc ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg
     3601 ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa
     3661 tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca
     3721 acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg
     3781 ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca
     3841 cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc
     3901 cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca
     3961 tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac
     4021 agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg
     4081 gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg
     4141 gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagg
     4201 atccgcgaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     4261 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     4321 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     4381 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     4441 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     4501 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     4561 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     4621 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcgcggat cctgcctcgc
     4681 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     4741 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     4801 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     4861 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     4921 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     4981 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5041 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5101 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5161 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5221 agataccagg gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5281 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5341 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5401 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5461 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5521 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     5581 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5641 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5701 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5761 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5821 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5881 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5941 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6001 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6061 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6121 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6181 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     6241 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6301 tattgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6361 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6421 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6481 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     6541 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6601 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6661 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6721 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6781 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6841 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     6901 ccattattat catgacat
//
