LOCUS       sequence-232692-        5666 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 5666)
  TITLE     SGC Toronto Q3-Q4 2017, Q1-Q2 2018
REFERENCE   2  (bases 1 to 5666)
  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: 6DXH (USP05)
FEATURES             Location/Qualifiers
     source          1..5666
                     /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"
     protein_bind    368..379
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     CDS             484..501
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(554..572)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      568..615
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      652..1107
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(739..758)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     949..970
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1199..2014)
                     /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(1199..2014)
                     /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     1922..1941
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2006..2025
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2136..2724
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2625..2644
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2878..2895
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2910..3049
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2975..2994)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3135..3157
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3151..3342)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4151..5233)
                     /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     5195..5214
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5234..5311)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5517..5536
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5620..5639
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5620..5638
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5639..5663
                     /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 gggcggggaa gtacggcagg tgtctaagca
      121 tgccttcagc ctcaagcagt tggacaaccc tgctcgaatc cctccctgtg gctggaagtg
      181 ctccaagtgt gacatgagag agaacctgtg gctcaacctg actgatggct ccatcctctg
      241 tgggcgacgc tacttcgatg gcagtggggg caacaaccac gctgtggagc actaccgaga
      301 gacaggctac ccgttagctg tcaagctggg caccatcacc cctgatggag ctgacgtgta
      361 ctcatatgat gaggatgaca tggtcctgga ccccagcctg gctgagcacc tgtcccactt
      421 cggcatcgac atgctgaaga tgcagaagac agactgatga cgaagcttgc ggccgcactc
      481 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
      541 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      601 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
      661 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
      721 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
      781 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
      841 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
      901 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
      961 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1021 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1081 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     1141 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     1201 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     1261 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     1321 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     1381 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     1441 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     1501 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     1561 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     1621 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     1681 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     1741 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     1801 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     1861 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     1921 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     1981 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     2041 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     2101 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2161 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2221 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2281 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2341 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2401 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     2461 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     2521 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     2581 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     2641 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     2701 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     2761 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     2821 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     2881 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     2941 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3001 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3061 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     3121 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     3181 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     3241 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     3301 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     3361 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     3421 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     3481 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     3541 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     3601 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     3661 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     3721 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     3781 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     3841 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     3901 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     3961 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     4021 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4081 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4141 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4201 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4261 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4321 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4381 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     4441 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     4501 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     4561 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     4621 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     4681 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     4741 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     4801 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     4861 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     4921 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     4981 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     5041 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     5101 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5161 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5221 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     5281 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     5341 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     5401 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     5461 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     5521 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     5581 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
     5641 aattgtgagc ggataacaat tcccct
//
