LOCUS       sequence-191796-        7047 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of human SH3 domain construct 
            MIA2-1/1-SV2/2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7047)
  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 7047)
  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-SV2/2
FEATURES             Location/Qualifiers
     source          1..7047
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     17..35
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        58..135
                     /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             136..1218
                     /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(155..174)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1461..1466,1467..1482,1483..1489)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1497..1513
                     /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     1502..1524
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1598..1615
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1631..2284
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2242..2264
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2291..2314
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2327..2344
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2702..2749
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(4342..4797)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4479..4500)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4691..4710
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4839..4861)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4947..5087
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5002..5021
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5102..5119)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5273..5860)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6031..6822,6823..6891))
                     /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     6654..6673
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6892..6996)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag aattcccgac
       61 accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
      121 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
      181 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
      241 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
      301 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
      361 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
      421 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
      481 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
      541 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
      601 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
      661 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
      721 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
      781 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
      841 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
      901 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat
      961 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg
     1021 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     1081 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa
     1141 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     1201 ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcaca
     1261 attctcatgt ttgacagctt atcatcgact gcacggtgca ccaatgcttc tggcgtcagg
     1321 cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg
     1381 ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa cggttctggc
     1441 aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt gtggaattgt
     1501 gagcggataa caatttcaca caggaaacag ccagtccgtt taggtgtttt cacgagcact
     1561 tcaccaacaa ggaccataga ttatgaaaat cgaagaacac catcaccatc accattccag
     1621 cggtaagctt atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac
     1681 tcgacttctt ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga
     1741 aggtgataaa tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta
     1801 ttatattgat ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga
     1861 caagcacaac atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg
     1921 agcggttttg gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac
     1981 tctcaaagtt gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt
     2041 atgtcataaa acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga
     2101 cgctcttgat gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt
     2161 ttgttttaaa aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa
     2221 gtatatagca tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc
     2281 aaaatcggat ctggaagttc tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg
     2341 ttccggtacc gcggcccagc cggccctgga atcaacgaaa ctgctggcag acctgaaaaa
     2401 atgtggtgac ctggaatgcg aagccctgat caatcgtgtg tcggcaatgc gtgattatcg
     2461 tggcccggac tgccgctacc tgaactttac caaaggtgaa gaaattagcg tgtatgttaa
     2521 actggcgggc gaacgtgaag atctgtgggc cggcaagggt aaagaatttg gttacttccc
     2581 gcgcgacgcg gtccagatcg aagaagtgtt cattagtgaa gaaatccaaa tgagcaccaa
     2641 agaatctgat tttctgtgtc tgctgggcgt ttcctatacg ttcgacgcgg ccgcataata
     2701 aaccgataca attaaaggct ccttttggag cctttttttt ggagattttc aacgtgaaaa
     2761 aattattatt cgcaattcct ttagttgttc ctttctattc tcactccgct gaaactgttg
     2821 aaagttgttt agcaaaaccc catacagaaa attcatttac taacgtctgg aaagacgaca
     2881 aaactttaga tcgttacgct aactatgagg gctgtctgtg gaatgctaca ggcgttgtag
     2941 tttgtactgg tgacgaaact cagtgtctag ctagagtggc ggtggctctg gttccggtga
     3001 ttttgattat gaaaagatgg caaacgctaa taagggggct atgaccgaaa atgccgatga
     3061 aaacgcgcta cagtctgacg ctaaaggcaa acttgattct gtcgctactg attacggtgc
     3121 tgctatcgat ggtttcattg gtgacgtttc cggccttgct aatggtaatg gtgctactgg
     3181 tgattttgct ggctctaatt cccaaatggc tcaagtcggt gacggtgata attcaccttt
     3241 aatgaataat ttccgtcaat atttaccttc cctccctcaa tcggttgaat gtcgcccttt
     3301 tgtctttggc gctggtaaac catatgaatt ttctattgat tgtgacaaaa taaacttatt
     3361 ccgtggtgtc tttgcgtttc ttttatatgt tgccaccttt atgtatgtat tttctacgtt
     3421 tgctaacata ctgcgtaata aggagtctta atcatgccag ttcttttggc tagcgccgcc
     3481 ctataccttg tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc
     3541 tgaatggaag ccggcggcac ctcgctaacg gattcaccac tccaagaatt ggagccaatc
     3601 aattcttgcg gagaactgtg aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt
     3661 ccgccatctc cagcagccgc acgcggcgca tctcgggcag cgttgggtcc tggccacggg
     3721 tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg
     3781 gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt
     3841 ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa
     3901 cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc
     3961 taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt
     4021 ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc
     4081 gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca
     4141 ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa
     4201 ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca
     4261 acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg
     4321 agctttaccg caggatccgc gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     4381 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     4441 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     4501 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     4561 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     4621 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     4681 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     4741 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcgc
     4801 ggatcctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     4861 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     4921 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     4981 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     5041 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     5101 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5161 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5221 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5281 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5341 cgacaggact ataaagatac cagggtttcc ccctggaagc tccctcgtgc gctctcctgt
     5401 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5461 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5521 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5581 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5641 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5701 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5761 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5821 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5881 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5941 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6001 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6061 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6121 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6181 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6241 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6301 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt
     6361 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6421 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6481 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6541 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6601 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac
     6661 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6721 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6781 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6841 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6901 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6961 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7021 tgacgtctaa gaaaccatta ttatcat
//
