LOCUS       sequence-232626-        6290 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 6290)
  TITLE     SGC Toronto Q3-Q4 2017, Q1-Q2 2018
REFERENCE   2  (bases 1 to 6290)
  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: 6BY9 (EHMT1)
FEATURES             Location/Qualifiers
     source          1..6290
                     /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             1108..1125
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1178..1196)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1192..1239
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1276..1731
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1363..1382)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1573..1594
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1823..2638)
                     /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(1823..2638)
                     /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     2546..2565
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2630..2649
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2760..3348
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3249..3268
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3502..3519
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3534..3673
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3599..3618)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3759..3781
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3775..3966)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4775..5857)
                     /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     5819..5838
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5858..5935)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6141..6160
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6244..6263
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6244..6262
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6263..6287
                     /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 gggcggcagt gctgccgggc caccactctc
      121 ggaggacgac aagctgcagg gtgcagcctc ccacgtgccc gagggctttg atccaacggg
      181 acctgctggg cttgggaggc caactcccgg cctttcccag ggaccaggga aggaaacctt
      241 ggagagcgct ctcatcgccc tcgactcgga aaaacccaag aagcttcgct tccacccaaa
      301 gcagctgtac ttctccgcca ggcaagggga gcttcagaag gtgctcctca tgctggtgga
      361 cggaattgac cccaacttca aaatggagca ccagaataag cgctctccac tgcacgccgc
      421 ggcagaggct ggacacgtgg acatctgcca catgctggtt caggcgggcg ctaatattga
      481 cacctgctca gaagaccaga ggaccccgtt gatggaagca gccgaaaaca accatctgga
      541 agcagtgaag tacctcatca aggctggggc cctggtggat cccaaggacg cagagggctc
      601 tacgtgtttg cacctggctg ccaagaaagg ccactacgaa gtggtccagt acctgctttc
      661 aaatggacag atggacgtca actgtcagga tgacggaggc tggacaccca tgatctgggc
      721 cacagagtac aagcacgtgg acctcgtgaa gctgctgctg tccaagggct ctgacatcaa
      781 catccgagac aacgaggaga acatttgcct gcactgggcg gcgttctccg gctgcgtgga
      841 catagccgag atcctgctgg ctgccaagtg cgacctccac gccgtgaaca tccacggaga
      901 ctcgccactg cacattgccg cccgggagaa ccgctacgac tgtgtcgtcc tctttctttc
      961 tcgggattca gatgtcacct taaagaacaa ggaaggagag acgcccctgc agtgtgcgag
     1021 cctcaactct caggtgtgga gcgctctgca gatgagcaag gctctgcagg actcggcctg
     1081 atgacgaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct
     1141 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1201 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1261 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     1321 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     1381 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     1441 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     1501 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1561 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1621 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1681 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     1741 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1801 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     1861 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     1921 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     1981 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     2041 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     2101 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     2161 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     2221 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     2281 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     2341 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     2401 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     2461 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     2521 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     2581 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     2641 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     2701 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2761 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2821 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2881 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     2941 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3001 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3061 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3121 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3181 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3241 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3301 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3361 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3421 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3481 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     3541 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     3601 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     3661 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3721 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     3781 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     3841 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     3901 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     3961 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     4021 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     4081 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     4141 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     4201 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     4261 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     4321 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     4381 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     4441 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     4501 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     4561 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     4621 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     4681 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     4741 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4801 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4861 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     4921 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     4981 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     5041 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     5101 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     5161 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     5221 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     5281 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     5341 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     5401 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     5461 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     5521 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     5581 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     5641 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     5701 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     5761 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     5821 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     5881 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     5941 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     6001 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     6061 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     6121 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     6181 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     6241 aattaatacg actcactata ggggaattgt gagcggataa caattcccct
//
