LOCUS       sequence-191127-        6939 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            SH3D21-2/3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6939)
  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 6939)
  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_SH3D21-2/3
FEATURES             Location/Qualifiers
     source          1..6939
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..39
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        62..139
                     /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             140..1222
                     /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(159..178)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1465..1470,1471..1486,1487..1493)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1501..1517
                     /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     1506..1528
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1602..1619
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1635..2288
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2246..2268
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2295..2318
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2331..2348
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2598..2645
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4238..4693)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4375..4396)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4587..4606
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4735..4757)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4843..4983
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4898..4917
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4998..5015)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5169..5756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5927..6718,6719..6787))
                     /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     6550..6569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6788..6892)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt caagaattcc
       61 cgacaccatc gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
      121 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
      181 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
      241 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
      301 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
      361 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
      421 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
      481 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
      541 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
      601 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
      661 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
      721 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
      781 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
      841 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
      901 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
      961 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     1021 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     1081 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     1141 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     1201 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     1261 cacaattctc atgtttgaca gcttatcatc gactgcacgg tgcaccaatg cttctggcgt
     1321 caggcagcca tcggaagctg tggtatggct gtgcaggtcg taaatcactg cataattcgt
     1381 gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc ataacggttc
     1441 tggcaaatat tctgaaatga gctgttgaca attaatcatc ggctcgtata atgtgtggaa
     1501 ttgtgagcgg ataacaattt cacacaggaa acagccagtc cgtttaggtg ttttcacgag
     1561 cacttcacca acaaggacca tagattatga aaatcgaaga acaccatcac catcaccatt
     1621 ccagcggtaa gcttatgtcc cctatactag gttattggaa aattaagggc cttgtgcaac
     1681 ccactcgact tcttttggaa tatcttgaag aaaaatatga agagcatttg tatgagcgcg
     1741 atgaaggtga taaatggcga aacaaaaagt ttgaattggg tttggagttt cccaatcttc
     1801 cttattatat tgatggtgat gttaaattaa cacagtctat ggccatcata cgttatatag
     1861 ctgacaagca caacatgttg ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg
     1921 aaggagcggt tttggatatt agatacggtg tttcgagaat tgcatatagt aaagactttg
     1981 aaactctcaa agttgatttt cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc
     2041 gtttatgtca taaaacatat ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt
     2101 atgacgctct tgatgttgtt ttatacatgg acccaatgtg cctggatgcg ttcccaaaat
     2161 tagtttgttt taaaaaacgt attgaagcta tcccacaaat tgataagtac ttgaaatcca
     2221 gcaagtatat agcatggcct ttgcagggct ggcaagccac gtttggtggt ggcgaccatc
     2281 ctccaaaatc ggatctggaa gttctgttcc aggggcccct gtccagcggt ctggttccgc
     2341 gtggttccgg taccgcggcc cagccggccc cggcaaaaca tccgcgcccg cagcgttggt
     2401 gtaaagtgaa tttctcctac tccccggaac aagcagacga actgaaactg caagcaggcg
     2461 aaatcgtgga aatgatcaaa gaaatcgaag atggttggtg gctgggcaag aaaaacggcc
     2521 agctgggtgc gtttccgagt aatttcgttg aactgctgga cagcggcccg ccgtctctgg
     2581 gtgcggccgc ataataaacc gatacaatta aaggctcctt ttggagcctt ttttttggag
     2641 attttcaacg tgaaaaaatt attattcgca attcctttag ttgttccttt ctattctcac
     2701 tccgctgaaa ctgttgaaag ttgtttagca aaaccccata cagaaaattc atttactaac
     2761 gtctggaaag acgacaaaac tttagatcgt tacgctaact atgagggctg tctgtggaat
     2821 gctacaggcg ttgtagtttg tactggtgac gaaactcagt gtctagctag agtggcggtg
     2881 gctctggttc cggtgatttt gattatgaaa agatggcaaa cgctaataag ggggctatga
     2941 ccgaaaatgc cgatgaaaac gcgctacagt ctgacgctaa aggcaaactt gattctgtcg
     3001 ctactgatta cggtgctgct atcgatggtt tcattggtga cgtttccggc cttgctaatg
     3061 gtaatggtgc tactggtgat tttgctggct ctaattccca aatggctcaa gtcggtgacg
     3121 gtgataattc acctttaatg aataatttcc gtcaatattt accttccctc cctcaatcgg
     3181 ttgaatgtcg cccttttgtc tttggcgctg gtaaaccata tgaattttct attgattgtg
     3241 acaaaataaa cttattccgt ggtgtctttg cgtttctttt atatgttgcc acctttatgt
     3301 atgtattttc tacgtttgct aacatactgc gtaataagga gtcttaatca tgccagttct
     3361 tttggctagc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag
     3421 ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt caccactcca
     3481 agaattggag ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa cccttggcag
     3541 aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc gggcagcgtt
     3601 gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg
     3661 cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac
     3721 tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt
     3781 tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc
     3841 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat
     3901 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca
     3961 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct
     4021 cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg
     4081 accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg
     4141 cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt
     4201 cacgaccacg ctgatgagct ttaccgcagg atccgcgaaa ttgtaagcgt taatattttg
     4261 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     4321 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     4381 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     4441 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     4501 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     4561 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     4621 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     4681 ctacagggcg cgtcgcggat cctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     4741 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     4801 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     4861 acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
     4921 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     4981 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5041 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5101 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5161 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5221 cagaggtggc gaaacccgac aggactataa agataccagg gtttccccct ggaagctccc
     5281 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5341 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5401 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5461 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5521 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5581 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     5641 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5701 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5761 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5821 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5881 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5941 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6001 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6061 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6121 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6181 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6241 ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6301 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6361 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6421 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6481 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6541 caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6601 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6661 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6721 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6781 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6841 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6901 cccgaaaagt gccacctgac gtctaagaaa ccattatta
//
