LOCUS       sequence-192390-        6927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            ITSN2-4/5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6927)
  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 6927)
  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_ITSN2-4/5
FEATURES             Location/Qualifiers
     source          1..6927
                     /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      2569..2616
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4209..4664)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4346..4367)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4558..4577
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4706..4728)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4814..4954
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4869..4888
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4969..4986)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5140..5727)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5898..6689,6690..6758))
                     /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     6521..6540
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6759..6863)
                     /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 ccatggccca gggcgcgctg gaaattgccc aggttacctc tgcctatgtt
     2401 gcatctggtt ccgaacaact gtctctggca ccaggtcaac tgattctgat tctgaaaaaa
     2461 aacacctccg gttggtggca aggtgaactg caagcccgtg gtaaaaaacg tcagaaaggt
     2521 tggtttccag cctcccatgt taaactgctg ggtgcggccg cataataaac cgatacaatt
     2581 aaaggctcct tttggagcct tttttttgga gattttcaac gtgaaaaaat tattattcgc
     2641 aattccttta gttgttcctt tctattctca ctccgctgaa actgttgaaa gttgtttagc
     2701 aaaaccccat acagaaaatt catttactaa cgtctggaaa gacgacaaaa ctttagatcg
     2761 ttacgctaac tatgagggct gtctgtggaa tgctacaggc gttgtagttt gtactggtga
     2821 cgaaactcag tgtctagcta gagtggcggt ggctctggtt ccggtgattt tgattatgaa
     2881 aagatggcaa acgctaataa gggggctatg accgaaaatg ccgatgaaaa cgcgctacag
     2941 tctgacgcta aaggcaaact tgattctgtc gctactgatt acggtgctgc tatcgatggt
     3001 ttcattggtg acgtttccgg ccttgctaat ggtaatggtg ctactggtga ttttgctggc
     3061 tctaattccc aaatggctca agtcggtgac ggtgataatt cacctttaat gaataatttc
     3121 cgtcaatatt taccttccct ccctcaatcg gttgaatgtc gcccttttgt ctttggcgct
     3181 ggtaaaccat atgaattttc tattgattgt gacaaaataa acttattccg tggtgtcttt
     3241 gcgtttcttt tatatgttgc cacctttatg tatgtatttt ctacgtttgc taacatactg
     3301 cgtaataagg agtcttaatc atgccagttc ttttggctag cgccgcccta taccttgtct
     3361 gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga atggaagccg
     3421 gcggcacctc gctaacggat tcaccactcc aagaattgga gccaatcaat tcttgcggag
     3481 aactgtgaat gcgcaaacca acccttggca gaacatatcc atcgcgtccg ccatctccag
     3541 cagccgcacg cggcgcatct cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcgt
     3601 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga
     3661 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc
     3721 aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
     3781 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac
     3841 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg
     3901 ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc
     3961 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa
     4021 cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat
     4081 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
     4141 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
     4201 gatccgcgaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     4261 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     4321 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     4381 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     4441 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     4501 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     4561 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     4621 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcgcgga tcctgcctcg
     4681 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     4741 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     4801 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct
     4861 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     4921 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
     4981 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5041 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5101 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5161 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5221 aagataccag ggtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5281 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5341 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5401 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5461 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5521 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     5581 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5641 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5701 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5761 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5821 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5881 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5941 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6001 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6061 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6121 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6181 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
     6241 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6301 atattgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6361 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6421 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6481 atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc
     6541 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6601 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6661 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6721 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6781 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6841 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     6901 accattatta tcatgacatt aacctat
//
