LOCUS       sequence-232684-        5804 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 5804)
  TITLE     SGC Toronto Q3-Q4 2017, Q1-Q2 2018
REFERENCE   2  (bases 1 to 5804)
  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: 6CDG (GID4)
FEATURES             Location/Qualifiers
     source          1..5804
                     /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"
     CDS             622..639
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(692..710)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      706..753
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      790..1245
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(877..896)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1087..1108
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1337..2152)
                     /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(1337..2152)
                     /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     2060..2079
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2144..2163
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2274..2862
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2763..2782
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3016..3033
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3048..3187
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3113..3132)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3273..3295
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3289..3480)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4289..5371)
                     /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     5333..5352
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5372..5449)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5655..5674
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5758..5777
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5758..5776
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5777..5801
                     /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 gggcagcggc tccaagttcc gcggccacca
      121 gaagagcaag gggaactcgt acgacgtaga ggtggtgctg cagcacgtgg acacgggaaa
      181 ctcttacctt tgtgggtact tgaagattaa aggccttact gaggagtatc caacccttac
      241 aaccttcttc gaaggagaaa taatcagcaa aaaacaccct ttcttaactc gcaagtggga
      301 tgcagatgaa gatgttgatc ggaaacactg gggcaagttt ctggcttttt atcagtatgc
      361 aaaatcattt aactcagatg actttgatta tgaagagctg aagaatggag actacgtctt
      421 catgaggtgg aaggaacagt ttctggtccc agatcacacg atcaaagaca tcagtggtgc
      481 ttcttttgcc gggttctact acatctgctt tcagaagtca gcagcctcca tagagggcta
      541 ctactaccat aggagttcag aatggtatca gtccctcaat ctaacccatg tttgatgacg
      601 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
      661 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
      721 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
      781 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
      841 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
      901 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
      961 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     1021 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1081 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1141 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     1201 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     1261 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1321 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     1381 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     1441 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     1501 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     1561 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     1621 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     1681 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     1741 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     1801 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     1861 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     1921 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     1981 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     2041 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     2101 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     2161 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     2221 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2281 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2341 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2401 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     2461 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2521 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2581 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2641 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     2701 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     2761 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     2821 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2881 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     2941 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3001 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     3061 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     3121 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     3181 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     3241 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     3301 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     3361 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     3421 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     3481 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     3541 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     3601 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     3661 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     3721 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     3781 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     3841 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     3901 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     3961 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     4021 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     4081 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     4141 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     4201 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     4261 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4321 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4381 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     4441 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     4501 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     4561 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     4621 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     4681 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     4741 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     4801 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     4861 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     4921 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     4981 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     5041 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     5101 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     5161 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     5221 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     5281 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     5341 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     5401 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     5461 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     5521 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     5581 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     5641 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     5701 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     5761 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccct
//
