LOCUS       sequence-319826-        5879 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 5879)
  TITLE     SGC Toronto 2019-Q1 to 2020-Q2
REFERENCE   2  (bases 1 to 5879)
  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: 6NM4
FEATURES             Location/Qualifiers
     source          1..5879
                     /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             744..761
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(814..832)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      828..875
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      912..1367
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(999..1018)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1209..1230
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1459..2274)
                     /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(1459..2274)
                     /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     2182..2201
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2266..2285
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2396..2984
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2885..2904
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3138..3155
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3170..3309
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3235..3254)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3395..3417
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3411..3602)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4411..5493)
                     /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     5455..5474
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5494..5571)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5777..5796
                     /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 cagcgagccg caggatgatg attacctcta ttgtgagatg
      181 tgtcagaact tcttcattga cagctgtgct gcccatgggc cccctacatt tgtaaaggac
      241 agtgcagtgg acaaggggca ccccaaccgt tcagccctca gtctgccccc agggctgaga
      301 attgggccat caggcatccc tcaggctggg cttggagtat ggaatgaggc atctgatctg
      361 ccgctgggtc tgcactttgg cccttatgag ggccgaatta cagaagacga agaggcagcc
      421 aacaatggat actcctggct gatcaccaag gggagaaact gctatgagta tgtggatgga
      481 aaagataaat cctgggccaa ctggatgagg tatgtgaact gtgcccggga tgatgaagag
      541 cagaacctgg tggccttcca gtaccacagg cagatcttct atagaacctg ccgagtcatt
      601 aggccaggct gtgaactgct ggtctggtat ggggatgaat acggccagga actgggcatc
      661 aagtggggca gcaagtggaa gaaagagctc atggcaggga gagaaccaaa gccatgatga
      721 cgaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
      781 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
      841 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
      901 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
      961 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1021 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1081 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1141 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1201 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     1261 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     1321 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     1381 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1441 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     1501 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     1561 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     1621 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     1681 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     1741 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     1801 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     1861 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1921 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1981 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     2041 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     2101 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     2161 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     2221 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     2281 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     2341 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2401 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2461 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2521 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2581 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2641 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2701 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2761 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2821 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2881 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2941 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3001 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3061 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3121 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     3181 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     3241 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3301 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3361 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3421 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     3481 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3541 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3601 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     3661 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     3721 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3781 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     3841 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3901 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3961 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     4021 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     4081 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     4141 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     4201 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     4261 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     4321 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4381 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4441 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4501 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4561 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4621 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4681 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4741 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4801 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     4861 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4921 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4981 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     5041 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5101 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5161 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5221 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     5281 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5341 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5401 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5461 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5521 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5581 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     5641 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     5701 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     5761 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     5821 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaat
//
