LOCUS       sequence-193491-        5579 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 5579)
  TITLE     SGC Toronto Q4 2016, Q1 Q2 2017
REFERENCE   2  (bases 1 to 5579)
  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: 5VBD (USP09X)
FEATURES             Location/Qualifiers
     source          1..5579
                     /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             444..461
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(514..532)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      528..575
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      612..1067
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(699..718)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     909..930
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1159..1974)
                     /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(1159..1974)
                     /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     1882..1901
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1966..1985
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2096..2684
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2585..2604
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2838..2855
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2870..3009
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2935..2954)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3095..3117
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3111..3302)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4111..5193)
                     /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     5155..5174
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5194..5271)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5477..5496
                     /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 cgcattccgc ggtaaacacc tctcttttgt agttcgattt
      181 ccaaaccagg gcagacaggt tgatgacttg gaggtatggt ctcatacaaa tgatacaatt
      241 ggttcagtac gacgatgtat tctcaatcgt attaaagcca acgtagccca tacaaaaatt
      301 gagctctttg tgggcggtga gctgatagat cctgcagatg atagaaagtt gattggacaa
      361 ttaaacttaa aagataaatc gcttattaca gccaaactta cacagataag ttcctgatga
      421 cgaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
      481 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
      541 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
      601 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
      661 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      721 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      781 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      841 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      901 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      961 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     1021 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     1081 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1141 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     1201 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     1261 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     1321 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     1381 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     1441 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     1501 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     1561 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1621 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1681 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1741 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1801 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1861 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1921 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     1981 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     2041 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2101 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2161 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2221 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2281 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2341 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2401 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     2461 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     2521 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2581 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2641 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2701 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2761 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2821 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2881 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     2941 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3001 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3061 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3121 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     3181 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3241 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3301 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     3361 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     3421 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     3481 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     3541 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3601 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3661 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3721 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     3781 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     3841 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     3901 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     3961 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     4021 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4081 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4141 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4201 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4261 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4321 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4381 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     4441 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     4501 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     4561 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4621 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4681 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4741 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4801 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4861 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4921 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4981 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5041 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5101 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5161 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5221 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5281 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     5341 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     5401 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     5461 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     5521 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaat
//
