LOCUS       sequence-319836-        6711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6711)
  TITLE     SGC Toronto 2019-Q1 to 2020-Q2
REFERENCE   2  (bases 1 to 6711)
  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: 6XKC
FEATURES             Location/Qualifiers
     source          1..6711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1291..1482
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1476..1498)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1584..1723
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1639..1658
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1738..1755)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1909..2497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1989..2008)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2608..2627)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2619..3434
                     /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             2619..3434
                     /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     complement(2692..2711)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3526..3981)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3663..3684)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3875..3894
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4018..4065)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4061..4079
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4132..4149)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4911..4928)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(4938..4955)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4983..5636)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     RBS             5644..5666
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5681..5705
                     /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     complement(5705..5724)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5706..5724)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5808..5827)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6033..6110
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(6111..6711,1..482)
                     /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(6130..6149)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
       61 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
      121 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
      181 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
      241 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
      301 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
      361 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
      421 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
      481 gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc tcgaccgatg
      541 cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc
      601 gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc
      661 tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt
      721 gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa
      781 cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc
      841 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
      901 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
      961 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     1021 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     1081 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     1141 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     1201 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     1261 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     1321 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     1381 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     1441 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     1501 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     1561 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     1621 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg
     1681 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     1741 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1801 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1861 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1921 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1981 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2041 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2101 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2161 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2221 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2281 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2341 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2401 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2461 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2521 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa
     2581 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa
     2641 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat
     2701 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg
     2761 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg
     2821 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta
     2881 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc
     2941 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc
     3001 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc
     3061 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg
     3121 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat
     3181 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg
     3241 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg
     3301 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt
     3361 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg
     3421 atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3481 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     3541 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     3601 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     3661 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     3721 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     3781 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     3841 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     3901 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     3961 atgcgccgct acagggcgcg tcccattcgc caatccggat atagttcctc ctttcagcaa
     4021 aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt gctcagcggt
     4081 ggcagcagcc aactcagctt cctttcgggc tttgttagca gccggatctc agtggtggtg
     4141 gtggtggtgc tcgagtgcgg ccgcaagctt cgtcatcaac gttctgtctt gctggtctgt
     4201 gcatggtgtg tcagaaaatc cacaatattt gtgtgaccag tcacacttgc tgagagaagg
     4261 ggagtcattc cataaccatc cttttccatc ttggcacaat acataagaag catcttcatg
     4321 atgtccaaac ttccagattc tgcacaatca tgcaatgcag tattaccttt gacacttttt
     4381 ctattaacat ctgccccctt ttcaagtaaa tactgagcaa tctctttatg tcctttgtaa
     4441 catgaaatca tcaagcacgt atgcccatgt cggtttgaca cttccaaatc agctttgtgt
     4501 tctacaaggt acttcactat ttccaaatgg ccatcgaaac acgcagctcg aagaggagtt
     4561 gaattggtta aagtcgtgtt gttgacagat gctccatgat ttaacaagga ctggaccacc
     4621 ttcagatgtc ctgctgcaga agcggcccat aaagggggag ccccctcaat ggtttcgcca
     4681 tcaaaattga cggagccccc aacttctatg gaggcactgc attgctctag gaggaattcc
     4741 accatgtcaa ggtgcccata cctggcggcc atcaagagtg gcgtggcccc atttgttttt
     4801 tcagagatca aggaggaaac ctcctctttg gatttgcttg ccaacaattt ggtgagaagc
     4861 cggagtttgc catcccgagc tgcgttaaat actgctgtct ttagatccat actaccacgc
     4921 ggaaccaggc cgctgctgtg atgatgatga tgatggctgc tgcccatgga tgaaccatcc
     4981 gattttggag gatggtcgcc accaccaaac gtggcttgcc agccctgcaa aggccatgct
     5041 atatacttgc tggatttcaa gtacttatca atttgtggga tagcttcaat acgtttttta
     5101 aaacaaacta attttgggaa cgcatccagg cacattgggt ccatgtataa aacaacatca
     5161 agagcgtcat acaacatgaa gtcaggatgg gttacatgat caccatttaa atatgtttta
     5221 tgacataaac gatcttcgaa cattttcagc atttcaggta gcttgctaag aaaatcaact
     5281 ttgagagttt caaagtcttt actatatgca attctcgaaa caccgtatct aatatccaaa
     5341 accgctcctt caagcattga aatctctgca cgctcttttg gacaaccacc caacatgttg
     5401 tgcttgtcag ctatataacg tatgatggcc atagactgtg ttaatttaac atcaccatca
     5461 atataataag gaagattggg aaactccaaa cccaattcaa actttttgtt tcgccattta
     5521 tcaccttcat cgcgctcata caaatgctct tcatattttt cttcaagata ttccaaaaga
     5581 agtcgagtgg gttgcacaag gcccttaatt ttccaataac ctagtatagg ggacatatgt
     5641 atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta tccgctcaca
     5701 attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc tctacgccgg
     5761 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
     5821 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
     5881 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc
     5941 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
     6001 ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc
     6061 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
     6121 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
     6181 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
     6241 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
     6301 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
     6361 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
     6421 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
     6481 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
     6541 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
     6601 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
     6661 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc t
//
