LOCUS       sequence-190997-        6894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            EPS8-1/1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6894)
  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 6894)
  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_EPS8-1/1
FEATURES             Location/Qualifiers
     source          1..6894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..40
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        63..140
                     /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             141..1223
                     /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(160..179)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1466..1471,1472..1487,1488..1494)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1502..1518
                     /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     1507..1529
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1603..1620
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1636..2289
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2247..2269
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2296..2319
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2332..2349
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2554..2601
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4194..4649)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4331..4352)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4543..4562
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4691..4713)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4799..4939
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4854..4873
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4954..4971)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5125..5712)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5883..6674,6675..6743))
                     /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     6506..6525
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6744..6848)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaattc
       61 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
      121 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      181 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      241 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      301 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      361 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      421 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      481 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      541 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      601 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      661 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      721 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      781 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      841 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      901 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
      961 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
     1021 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1081 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1141 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1201 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1261 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1321 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1381 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1441 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1501 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1561 gcacttcacc aacaaggacc atagattatg aaaatcgaag aacaccatca ccatcaccat
     1621 tccagcggta agcttatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
     1681 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
     1741 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
     1801 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
     1861 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
     1921 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
     1981 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
     2041 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
     2101 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
     2161 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
     2221 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
     2281 cctccaaaat cggatctgga agttctgttc caggggcccc tgtccagcgg tctggttccg
     2341 cgtggttccg gtaccgcggc ccagccggcc aaatacgcaa aatctaaata tgacttcgtc
     2401 gcccgcaata attctgaact gagcgtcctg aaagatgaca ttctggaaat cctggatgat
     2461 cgtaaacagt ggtggaaagt ccgcaacgcg agcggcgatt ctggttttgt gccgaacaat
     2521 attctggaca tcgttcgtgc ggccgcataa taaaccgata caattaaagg ctccttttgg
     2581 agcctttttt ttggagattt tcaacgtgaa aaaattatta ttcgcaattc ctttagttgt
     2641 tcctttctat tctcactccg ctgaaactgt tgaaagttgt ttagcaaaac cccatacaga
     2701 aaattcattt actaacgtct ggaaagacga caaaacttta gatcgttacg ctaactatga
     2761 gggctgtctg tggaatgcta caggcgttgt agtttgtact ggtgacgaaa ctcagtgtct
     2821 agctagagtg gcggtggctc tggttccggt gattttgatt atgaaaagat ggcaaacgct
     2881 aataaggggg ctatgaccga aaatgccgat gaaaacgcgc tacagtctga cgctaaaggc
     2941 aaacttgatt ctgtcgctac tgattacggt gctgctatcg atggtttcat tggtgacgtt
     3001 tccggccttg ctaatggtaa tggtgctact ggtgattttg ctggctctaa ttcccaaatg
     3061 gctcaagtcg gtgacggtga taattcacct ttaatgaata atttccgtca atatttacct
     3121 tccctccctc aatcggttga atgtcgccct tttgtctttg gcgctggtaa accatatgaa
     3181 ttttctattg attgtgacaa aataaactta ttccgtggtg tctttgcgtt tcttttatat
     3241 gttgccacct ttatgtatgt attttctacg tttgctaaca tactgcgtaa taaggagtct
     3301 taatcatgcc agttcttttg gctagcgccg ccctatacct tgtctgcctc cccgcgttgc
     3361 gtcgcggtgc atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa
     3421 cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca
     3481 aaccaaccct tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg
     3541 catctcgggc agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     3601 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     3661 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     3721 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     3781 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     3841 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     3901 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     3961 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     4021 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     4081 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     4141 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcaggatcc gcgaaattgt
     4201 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     4261 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     4321 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     4381 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     4441 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     4501 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     4561 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     4621 cgcgcttaat gcgccgctac agggcgcgtc gcggatcctg cctcgcgcgt ttcggtgatg
     4681 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4741 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     4801 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     4861 agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
     4921 gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4981 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5041 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5101 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5161 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accagggttt
     5221 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5281 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5341 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5401 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5461 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5521 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     5581 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5641 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5701 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5761 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     5821 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     5881 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5941 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6001 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6061 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6121 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6181 aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6241 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6301 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6361 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6421 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6481 tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg
     6541 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6601 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6661 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6721 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     6781 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     6841 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tatt
//
