LOCUS       sequence-319835-        5483 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 5483)
  TITLE     SGC Toronto 2019-Q1 to 2020-Q2
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: 6WAU
FEATURES             Location/Qualifiers
     source          1..5483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             15..37
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             47..64
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             77..97
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             304..321
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(374..392)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      388..435
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      472..927
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(559..578)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     769..790
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1019..1834)
                     /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(1019..1834)
                     /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     1742..1761
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1826..1845
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1956..2544
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2445..2464
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2698..2715
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2730..2869
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2795..2814)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2955..2977
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2971..3162)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3971..5053)
                     /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     5015..5034
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5054..5131)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5337..5356
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5440..5459
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5440..5458
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5459..5483
                     /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 cctctagaaa taattttgtt taactttaag aaggagatat accatgcatc atcatcatca
       61 tcacagcagc ggcagagaaa acttgtattt ccagggctcc aaactgacgg agggccagta
      121 tgtgctgtgc cggtggacag atggcctgta ctacctcggg aagatcaaga gggtcagcag
      181 ctctaagcaa agctgcctcg tgactttcga agataattcc aaatactggg tcctatggaa
      241 ggacatacag catgccggtg ttccaggaga ggagtgatga cgaagcttgc ggccgcactc
      301 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
      361 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      421 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
      481 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      541 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      601 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
      661 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
      721 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
      781 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
      841 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
      901 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
      961 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     1021 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     1081 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     1141 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     1201 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     1261 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     1321 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     1381 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     1441 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     1501 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     1561 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     1621 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     1681 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     1741 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     1801 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     1861 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     1921 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     1981 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2041 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2101 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2161 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2221 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2281 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2341 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2401 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2461 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2521 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2581 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2641 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     2701 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     2761 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2821 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     2881 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     2941 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     3001 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     3061 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     3121 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     3181 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     3241 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     3301 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     3361 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     3421 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     3481 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     3541 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     3601 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     3661 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     3721 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     3781 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     3841 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     3901 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     3961 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4021 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4081 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4141 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4201 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     4261 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4321 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4381 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4441 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4501 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4561 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4621 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4681 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     4741 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     4801 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     4861 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     4921 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     4981 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5041 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     5101 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     5161 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     5221 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     5281 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     5341 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     5401 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
     5461 aattgtgagc ggataacaat tcc
//
