LOCUS       sequence-232690-        6210 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6210)
  TITLE     SGC Toronto Q3-Q4 2017, Q1-Q2 2018
REFERENCE   2  (bases 1 to 6210)
  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"
COMMENT     Sequence Label: 6D1T (MBD1)
FEATURES             Location/Qualifiers
     source          1..6210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             42..695
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             723..740
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             750..767
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1028..1045
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1098..1116)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1112..1159
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1196..1651
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1283..1302)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1493..1514
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1743..2558)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1743..2558)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2466..2485
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2550..2569
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2680..3268
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3169..3188
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3422..3439
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3454..3593
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3519..3538)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3679..3701
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3695..3886)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4695..5777)
                     /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     5739..5758
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5778..5855)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6061..6080
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6164..6183
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6164..6182
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6183..6207
                     /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)."
ORIGIN
        1 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgtcccct atactaggtt
       61 attggaaaat taagggcctt gtgcaaccca ctcgacttct tttggaatat cttgaagaaa
      121 aatatgaaga gcatttgtat gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg
      181 aattgggttt ggagtttccc aatcttcctt attatattga tggtgatgtt aaattaacac
      241 agtctatggc catcatacgt tatatagctg acaagcacaa catgttgggt ggttgtccaa
      301 aagagcgtgc agagatttca atgcttgaag gagcggtttt ggatattaga tacggtgttt
      361 cgagaattgc atatagtaaa gactttgaaa ctctcaaagt tgattttctt agcaagctac
      421 ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa aacatattta aatggtgatc
      481 atgtaaccca tcctgacttc atgttgtatg acgctcttga tgttgtttta tacatggacc
      541 caatgtgcct ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt gaagctatcc
      601 cacaaattga taagtacttg aaatccagca agtatatagc atggcctttg cagggctggc
      661 aagccacgtt tggtggtggc gaccatcctc caaaatcgga tggttcatcc atgggcagca
      721 gccatcatca tcatcatcac agcagcggcc tggttccgcg tggtagtatg gctgaggact
      781 ggctggactg cccggccctg ggccctggct ggaagcgccg cgaagtcttt cgcaagtcag
      841 gggccacctg tggacgctca gacacctatt accagagccc cacaggagac aggatccgaa
      901 gcaaagttga gctgactcga tacctgggcc ctgcgtgtga tctcaccctc ttcgacttca
      961 aacaaggcat cttgtgctat ccagccccca aggcccattg atgacgaagc ttgcggccgc
     1021 actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc
     1081 tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     1141 ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg
     1201 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     1261 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     1321 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     1381 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     1441 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     1501 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     1561 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     1621 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     1681 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat
     1741 tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     1801 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     1861 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     1921 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     1981 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     2041 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     2101 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     2161 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     2221 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     2281 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     2341 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     2401 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     2461 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     2521 ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     2581 atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca
     2641 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2701 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2761 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2821 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2881 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2941 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3001 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3061 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3121 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3181 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3241 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3301 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3361 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     3421 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     3481 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     3541 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3601 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3661 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3721 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     3781 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     3841 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     3901 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     3961 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     4021 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     4081 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     4141 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     4201 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     4261 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     4321 tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct
     4381 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     4441 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     4501 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     4561 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     4621 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     4681 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4741 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     4801 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     4861 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     4921 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     4981 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     5041 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     5101 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     5161 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     5221 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     5281 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     5341 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     5401 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     5461 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     5521 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     5581 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     5641 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     5701 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     5761 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     5821 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     5881 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     5941 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     6001 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     6061 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     6121 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     6181 ggggaattgt gagcggataa caattcccct
//
