LOCUS       sequence-192218-        6921 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            MIA2-1/1-SV1/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6921)
  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 6921)
  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_MIA2-1/1-SV1/2
FEATURES             Location/Qualifiers
     source          1..6921
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(717..722,723..738,739..745)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    753..769
                     /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     758..780
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             854..871
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             887..1540
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1498..1520
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1547..1570
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1583..1600
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      1832..1879
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3472..3927)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3609..3630)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3821..3840
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3969..3991)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4077..4217
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4132..4151
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4232..4249)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4403..4990)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5161..5952,5953..6021))
                     /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     5784..5803
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6022..6126)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6194..6212
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6235..6312
                     /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             join(6313..6921,1..474)
                     /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(6332..6351)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
       61 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
      121 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
      181 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
      241 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
      301 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
      361 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
      421 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
      481 cgcaattaat gtaagttagc tcactcatta ggcacaattc tcatgtttga cagcttatca
      541 tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc tgtggtatgg
      601 ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca aggcgcactc ccgttctgga
      661 taatgttttt tgcgccgaca tcataacggt tctggcaaat attctgaaat gagctgttga
      721 caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacacagg
      781 aaacagccag tccgtttagg tgttttcacg agcacttcac caacaaggac catagattat
      841 gaaaatcgaa gaacaccatc accatcacca ttccagcggt aagcttatgt cccctatact
      901 aggttattgg aaaattaagg gccttgtgca acccactcga cttcttttgg aatatcttga
      961 agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt gataaatggc gaaacaaaaa
     1021 gtttgaattg ggtttggagt ttcccaatct tccttattat attgatggtg atgttaaatt
     1081 aacacagtct atggccatca tacgttatat agctgacaag cacaacatgt tgggtggttg
     1141 tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg gttttggata ttagatacgg
     1201 tgtttcgaga attgcatata gtaaagactt tgaaactctc aaagttgatt ttcttagcaa
     1261 gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt cataaaacat atttaaatgg
     1321 tgatcatgta acccatcctg acttcatgtt gtatgacgct cttgatgttg ttttatacat
     1381 ggacccaatg tgcctggatg cgttcccaaa attagtttgt tttaaaaaac gtattgaagc
     1441 tatcccacaa attgataagt acttgaaatc cagcaagtat atagcatggc ctttgcaggg
     1501 ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa tcggatctgg aagttctgtt
     1561 ccaggggccc ctgtccagcg gtctggttcc gcgtggttcc ggtaccgcgg cccagccggc
     1621 catggcccag ggcgcgctga atcgtgtttc cgccatgcgt gattatcgtg gtccggattg
     1681 tcgttacctg aacttcacca aaggtgaaga aatctccgtc tacgttaaac tggcaggtga
     1741 acgtgaagat ttgtgggcag gttccaaagg taaagaattt ggttactttc cgcgtgatgc
     1801 cgttcagatt gaagaagcgg ccgcataata aaccgataca attaaaggct ccttttggag
     1861 cctttttttt ggagattttc aacgtgaaaa aattattatt cgcaattcct ttagttgttc
     1921 ctttctattc tcactccgct gaaactgttg aaagttgttt agcaaaaccc catacagaaa
     1981 attcatttac taacgtctgg aaagacgaca aaactttaga tcgttacgct aactatgagg
     2041 gctgtctgtg gaatgctaca ggcgttgtag tttgtactgg tgacgaaact cagtgtctag
     2101 ctagagtggc ggtggctctg gttccggtga ttttgattat gaaaagatgg caaacgctaa
     2161 taagggggct atgaccgaaa atgccgatga aaacgcgcta cagtctgacg ctaaaggcaa
     2221 acttgattct gtcgctactg attacggtgc tgctatcgat ggtttcattg gtgacgtttc
     2281 cggccttgct aatggtaatg gtgctactgg tgattttgct ggctctaatt cccaaatggc
     2341 tcaagtcggt gacggtgata attcaccttt aatgaataat ttccgtcaat atttaccttc
     2401 cctccctcaa tcggttgaat gtcgcccttt tgtctttggc gctggtaaac catatgaatt
     2461 ttctattgat tgtgacaaaa taaacttatt ccgtggtgtc tttgcgtttc ttttatatgt
     2521 tgccaccttt atgtatgtat tttctacgtt tgctaacata ctgcgtaata aggagtctta
     2581 atcatgccag ttcttttggc tagcgccgcc ctataccttg tctgcctccc cgcgttgcgt
     2641 cgcggtgcat ggagccgggc cacctcgacc tgaatggaag ccggcggcac ctcgctaacg
     2701 gattcaccac tccaagaatt ggagccaatc aattcttgcg gagaactgtg aatgcgcaaa
     2761 ccaacccttg gcagaacata tccatcgcgt ccgccatctc cagcagccgc acgcggcgca
     2821 tctcgggcag cgttgggtcc tggccacggg tgcgcatgat cgtgctcctg tcgttgagga
     2881 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
     2941 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
     3001 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
     3061 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     3121 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     3181 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     3241 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     3301 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     3361 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     3421 tctgtgaatc gcttcacgac cacgctgatg agctttaccg caggatccgc gaaattgtaa
     3481 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     3541 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     3601 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     3661 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     3721 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     3781 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     3841 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     3901 cgcttaatgc gccgctacag ggcgcgtcgc ggatcctgcc tcgcgcgttt cggtgatgac
     3961 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     4021 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     4081 gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag
     4141 agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga
     4201 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4261 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4321 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4381 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4441 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac cagggtttcc
     4501 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4561 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4621 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4681 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4741 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4801 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     4861 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4921 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4981 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5041 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5101 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5161 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5221 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5281 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5341 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5401 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5461 cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5521 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatattgt gcaaaaaagc
     5581 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5641 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5701 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5761 ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct
     5821 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5881 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     5941 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6001 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6061 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6121 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     6181 attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat tcccgacacc
     6241 atcgaatggt gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     6301 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     6361 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     6421 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     6481 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     6541 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     6601 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     6661 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     6721 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     6781 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     6841 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     6901 cgtctggctg gctggcataa a
//
