LOCUS       sequence-192296-        6918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            SASH3-1/1.
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_SASH3-1/1
FEATURES             Location/Qualifiers
     source          1..6918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..37
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        60..137
                     /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             138..1220
                     /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(157..176)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1463..1468,1469..1484,1485..1491)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1499..1515
                     /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     1504..1526
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1600..1617
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1633..2286
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2244..2266
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2293..2316
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2329..2346
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2575..2622
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4215..4670)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4352..4373)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4564..4583
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4712..4734)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4820..4960
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4875..4894
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4975..4992)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5146..5733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5904..6695,6696..6764))
                     /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     6527..6546
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6765..6869)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattcccg
       61 acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
      121 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
      181 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
      241 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
      301 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
      361 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
      421 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
      481 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
      541 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
      601 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
      661 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
      721 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
      781 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
      841 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
      901 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
      961 ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
     1021 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
     1081 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
     1141 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     1201 gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca
     1261 caattctcat gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca
     1321 ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt
     1381 cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg
     1441 gcaaatattc tgaaatgagc tgttgacaat taatcatcgg ctcgtataat gtgtggaatt
     1501 gtgagcggat aacaatttca cacaggaaac agccagtccg tttaggtgtt ttcacgagca
     1561 cttcaccaac aaggaccata gattatgaaa atcgaagaac accatcacca tcaccattcc
     1621 agcggtaagc ttatgtcccc tatactaggt tattggaaaa ttaagggcct tgtgcaaccc
     1681 actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta tgagcgcgat
     1741 gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc caatcttcct
     1801 tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg ttatatagct
     1861 gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc aatgcttgaa
     1921 ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa agactttgaa
     1981 actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt
     2041 ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt catgttgtat
     2101 gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt cccaaaatta
     2161 gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt gaaatccagc
     2221 aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg cgaccatcct
     2281 ccaaaatcgg atctggaagt tctgttccag gggcccctgt ccagcggtct ggttccgcgt
     2341 ggttccggta ccgcggccca gccggccatg gcccagggcg cgctgggtcg tgctcgtgtt
     2401 cataccgatt tcaccccatc cccgtatgat catgattccc tgaaactgca aaaaggtgat
     2461 gttattcaga ttatcgaaaa accgccagtt ggtacttggc tgggtctgct gaatggtaaa
     2521 gtcggttcct tcaaattcat ttacgttgat gtcctgccgg cggccgcata ataaaccgat
     2581 acaattaaag gctccttttg gagccttttt tttggagatt ttcaacgtga aaaaattatt
     2641 attcgcaatt cctttagttg ttcctttcta ttctcactcc gctgaaactg ttgaaagttg
     2701 tttagcaaaa ccccatacag aaaattcatt tactaacgtc tggaaagacg acaaaacttt
     2761 agatcgttac gctaactatg agggctgtct gtggaatgct acaggcgttg tagtttgtac
     2821 tggtgacgaa actcagtgtc tagctagagt ggcggtggct ctggttccgg tgattttgat
     2881 tatgaaaaga tggcaaacgc taataagggg gctatgaccg aaaatgccga tgaaaacgcg
     2941 ctacagtctg acgctaaagg caaacttgat tctgtcgcta ctgattacgg tgctgctatc
     3001 gatggtttca ttggtgacgt ttccggcctt gctaatggta atggtgctac tggtgatttt
     3061 gctggctcta attcccaaat ggctcaagtc ggtgacggtg ataattcacc tttaatgaat
     3121 aatttccgtc aatatttacc ttccctccct caatcggttg aatgtcgccc ttttgtcttt
     3181 ggcgctggta aaccatatga attttctatt gattgtgaca aaataaactt attccgtggt
     3241 gtctttgcgt ttcttttata tgttgccacc tttatgtatg tattttctac gtttgctaac
     3301 atactgcgta ataaggagtc ttaatcatgc cagttctttt ggctagcgcc gccctatacc
     3361 ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg ggccacctcg acctgaatgg
     3421 aagccggcgg cacctcgcta acggattcac cactccaaga attggagcca atcaattctt
     3481 gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg cgtccgccat
     3541 ctccagcagc cgcacgcggc gcatctcggg cagcgttggg tcctggccac gggtgcgcat
     3601 gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca
     3661 gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac
     3721 ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa
     3781 gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg
     3841 tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg
     3901 gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg
     3961 ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc
     4021 catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct
     4081 taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct
     4141 ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta
     4201 ccgcaggatc cgcgaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     4261 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     4321 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     4381 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     4441 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     4501 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     4561 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     4621 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cgcggatcct
     4681 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     4741 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     4801 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
     4861 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga
     4921 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct
     4981 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5041 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5101 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5161 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5221 actataaaga taccagggtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5281 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5341 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5401 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5461 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5521 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5581 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5641 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5701 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5761 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5821 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5881 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5941 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6001 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6061 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6121 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6181 tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc
     6241 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6301 tcccccatat tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6361 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6421 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6481 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
     6541 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6601 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6661 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6721 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6781 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6841 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     6901 taagaaacca ttattatc
//
