LOCUS       sequence-191799-        7077 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            MIA3-1/1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7077)
  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 7077)
  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_MIA3-1/1
FEATURES             Location/Qualifiers
     source          1..7077
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..48
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        71..148
                     /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             149..1231
                     /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(168..187)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1474..1479,1480..1495,1496..1502)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1510..1526
                     /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     1515..1537
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1611..1628
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1644..2297
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2255..2277
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2304..2327
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2340..2357
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2745..2792
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4385..4840)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4522..4543)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4734..4753
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4882..4904)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4990..5130
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5045..5064
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5145..5162)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5316..5903)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6074..6865,6866..6934))
                     /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     6697..6716
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6935..7039)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
       61 aagaattccc gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc
      121 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
      181 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
      241 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
      301 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
      361 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
      421 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
      481 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
      541 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
      601 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
      661 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
      721 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
      781 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
      841 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
      901 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
      961 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     1021 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     1081 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     1141 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     1201 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     1261 ctcattaggc acaattctca tgtttgacag cttatcatcg actgcacggt gcaccaatgc
     1321 ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc
     1381 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca
     1441 taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa
     1501 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagccagtcc gtttaggtgt
     1561 tttcacgagc acttcaccaa caaggaccat agattatgaa aatcgaagaa caccatcacc
     1621 atcaccattc cagcggtaag cttatgtccc ctatactagg ttattggaaa attaagggcc
     1681 ttgtgcaacc cactcgactt cttttggaat atcttgaaga aaaatatgaa gagcatttgt
     1741 atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc
     1801 ccaatcttcc ttattatatt gatggtgatg ttaaattaac acagtctatg gccatcatac
     1861 gttatatagc tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt gcagagattt
     1921 caatgcttga aggagcggtt ttggatatta gatacggtgt ttcgagaatt gcatatagta
     1981 aagactttga aactctcaaa gttgattttc ttagcaagct acctgaaatg ctgaaaatgt
     2041 tcgaagatcg tttatgtcat aaaacatatt taaatggtga tcatgtaacc catcctgact
     2101 tcatgttgta tgacgctctt gatgttgttt tatacatgga cccaatgtgc ctggatgcgt
     2161 tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat cccacaaatt gataagtact
     2221 tgaaatccag caagtatata gcatggcctt tgcagggctg gcaagccacg tttggtggtg
     2281 gcgaccatcc tccaaaatcg gatctggaag ttctgttcca ggggcccctg tccagcggtc
     2341 tggttccgcg tggttccggt accgcggccc agccggcccc gagtacgggt cgtcgtttct
     2401 ccgaacataa actgtgtgca gatgatgaat gctcaatgct gatgtatcgt ggtgaagctc
     2461 tggaagattt taccggcccg gactgccgtt ttgtcaactt caaaaaaggt gatccggtct
     2521 atgtgtatta caaactggca cgtggttggc cggaagtttg ggcaggtagc gtcggtcgca
     2581 cctttggtta tttcccgaaa gatctgattc aggtggttca tgaatacacg aaagaagaac
     2641 tgcaagtgcc gaccgatgaa acggactttg tttgtttcga cggcggtcgc gatgacttcc
     2701 acaactacaa tgttgaagaa ctgctgggcg cggccgcata ataaaccgat acaattaaag
     2761 gctccttttg gagccttttt tttggagatt ttcaacgtga aaaaattatt attcgcaatt
     2821 cctttagttg ttcctttcta ttctcactcc gctgaaactg ttgaaagttg tttagcaaaa
     2881 ccccatacag aaaattcatt tactaacgtc tggaaagacg acaaaacttt agatcgttac
     2941 gctaactatg agggctgtct gtggaatgct acaggcgttg tagtttgtac tggtgacgaa
     3001 actcagtgtc tagctagagt ggcggtggct ctggttccgg tgattttgat tatgaaaaga
     3061 tggcaaacgc taataagggg gctatgaccg aaaatgccga tgaaaacgcg ctacagtctg
     3121 acgctaaagg caaacttgat tctgtcgcta ctgattacgg tgctgctatc gatggtttca
     3181 ttggtgacgt ttccggcctt gctaatggta atggtgctac tggtgatttt gctggctcta
     3241 attcccaaat ggctcaagtc ggtgacggtg ataattcacc tttaatgaat aatttccgtc
     3301 aatatttacc ttccctccct caatcggttg aatgtcgccc ttttgtcttt ggcgctggta
     3361 aaccatatga attttctatt gattgtgaca aaataaactt attccgtggt gtctttgcgt
     3421 ttcttttata tgttgccacc tttatgtatg tattttctac gtttgctaac atactgcgta
     3481 ataaggagtc ttaatcatgc cagttctttt ggctagcgcc gccctatacc ttgtctgcct
     3541 ccccgcgttg cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg
     3601 cacctcgcta acggattcac cactccaaga attggagcca atcaattctt gcggagaact
     3661 gtgaatgcgc aaaccaaccc ttggcagaac atatccatcg cgtccgccat ctccagcagc
     3721 cgcacgcggc gcatctcggg cagcgttggg tcctggccac gggtgcgcat gatcgtgctc
     3781 ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca gaatgaatca
     3841 ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca
     3901 acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc
     3961 tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg tggaacacct
     4021 acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg gtcccgccgc
     4081 atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg ttcatcatca
     4141 gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc catgaacaga
     4201 aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct taacatggcc
     4261 cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat
     4321 gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcaggatc
     4381 cgcgaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     4441 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     4501 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     4561 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     4621 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     4681 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     4741 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     4801 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cgcggatcct gcctcgcgcg
     4861 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     4921 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     4981 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
     5041 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     5101 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     5161 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5221 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5281 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5341 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5401 taccagggtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5461 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5521 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5581 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5641 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5701 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     5761 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5821 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5881 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5941 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6001 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6061 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6121 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6181 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6241 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6301 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6361 atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg
     6421 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6481 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6541 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6601 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6661 ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca catagcagaa
     6721 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6781 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6841 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6901 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6961 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     7021 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaa
//
