LOCUS       sequence-232631-        5540 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 5540)
  TITLE     SGC Toronto Q3-Q4 2017, Q1-Q2 2018
REFERENCE   2  (bases 1 to 5540)
  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: 6C1A (MBD2)
FEATURES             Location/Qualifiers
     source          1..5540
                     /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             44..61
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             74..94
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             358..375
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(428..446)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      442..489
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      526..981
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(613..632)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     823..844
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1073..1888)
                     /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(1073..1888)
                     /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     1796..1815
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1880..1899
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2010..2598
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2499..2518
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2752..2769
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2784..2923
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2849..2868)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3009..3031
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3025..3216)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4025..5107)
                     /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     5069..5088
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5108..5185)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5391..5410
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5494..5513
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5494..5512
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5513..5537
                     /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 gagatatacc atgcatcatc atcatcatca
       61 cagcagcggc agagaaaact tgtatttcca gggcgccacg gagagcggga agaggatgga
      121 ttgcccggcc ctcccccccg gatggaagaa ggaggaagtg atccgaaaat ctgggctaag
      181 tgctggcaag agcgatgtct actacttcag tccaagtggt aagaagttca gaagcaagcc
      241 tcagttggca aggtacctgg gaaatactgt tgatctcagc agttttgact tcagaactgg
      301 aaagatgatg cctagtaaat tacagaagtg atgacgaagc ttgcggccgc actcgagcac
      361 caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      421 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      481 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
      541 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
      601 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
      661 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
      721 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
      781 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
      841 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
      901 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
      961 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     1021 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
     1081 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     1141 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     1201 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     1261 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     1321 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     1381 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     1441 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     1501 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     1561 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     1621 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     1681 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     1741 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     1801 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     1861 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     1921 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     1981 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2041 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2101 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     2161 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2221 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2281 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2341 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2401 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2461 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2521 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2581 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     2641 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     2701 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     2761 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     2821 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     2881 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     2941 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3001 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3061 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     3121 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     3181 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     3241 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     3301 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     3361 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     3421 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     3481 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     3541 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     3601 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     3661 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     3721 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     3781 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     3841 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     3901 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     3961 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     4021 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     4081 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     4141 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     4201 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     4261 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc
     4321 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     4381 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     4441 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     4501 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     4561 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     4621 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     4681 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     4741 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     4801 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     4861 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     4921 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     4981 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     5041 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     5101 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     5161 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     5221 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     5281 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     5341 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     5401 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     5461 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
     5521 gagcggataa caattcccct
//
