LOCUS       sequence-193498-        5483 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5483)
  TITLE     SGC Toronto Q4 2016, Q1 Q2 2017
REFERENCE   2  (bases 1 to 5483)
  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: 5W9Q (MBD1)
FEATURES             Location/Qualifiers
     source          1..5483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             91..108
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             121..141
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             348..365
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(418..436)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      432..479
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      516..971
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(603..622)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     813..834
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1063..1878)
                     /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(1063..1878)
                     /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     1786..1805
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1870..1889
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2000..2588
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2489..2508
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2742..2759
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2774..2913
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2839..2858)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2999..3021
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3015..3206)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4015..5097)
                     /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     5059..5078
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5098..5175)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5381..5400
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg catcatcatc atcatcacag cagcggcaga
      121 gaaaacttgt atttccaggg caccaaccgt cgccagaatc gtaaatgcgg tgcatgtgca
      181 gcatgcctgc gtcgcatgga ttgtggtcgc tgcgattttt gctgtgacaa accgaagttc
      241 ggcggtagca accagaaacg tcaaaagtgt cgttggcgcc agtgcctgca atttgcaatg
      301 aaacgcctgc tgccgtcatg atgacgaagc ttgcggccgc actcgagcac caccaccacc
      361 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
      421 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
      481 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
      541 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
      601 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
      661 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
      721 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
      781 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
      841 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
      901 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
      961 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     1021 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     1081 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     1141 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     1201 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     1261 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     1321 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     1381 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     1441 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     1501 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     1561 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     1621 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     1681 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     1741 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     1801 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     1861 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     1921 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1981 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2041 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2101 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     2161 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2221 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2281 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2341 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     2401 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     2461 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     2521 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     2581 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     2641 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     2701 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     2761 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     2821 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     2881 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     2941 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     3001 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     3061 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     3121 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     3181 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     3241 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     3301 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     3361 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     3421 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     3481 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     3541 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     3601 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     3661 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     3721 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     3781 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     3841 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     3901 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     3961 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     4021 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4081 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     4141 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     4201 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     4261 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     4321 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     4381 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     4441 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     4501 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     4561 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     4621 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     4681 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     4741 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     4801 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     4861 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     4921 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     4981 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     5041 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     5101 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     5161 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     5221 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     5281 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     5341 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     5401 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     5461 tcgagatctc gatcccgcga aat
//
