LOCUS       sequence-194721-        5507 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 5507)
  TITLE     SGC Toronto Q2 Q3 2016
REFERENCE   2  (bases 1 to 5507)
  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: 5T1I (CBX3)
FEATURES             Location/Qualifiers
     source          1..5507
                     /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             372..389
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(442..460)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      456..503
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      540..995
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(627..646)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     837..858
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1087..1902)
                     /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(1087..1902)
                     /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     1810..1829
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1894..1913
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2024..2612
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2513..2532
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2766..2783
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2798..2937
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2863..2882)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3023..3045
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3039..3230)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4039..5121)
                     /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     5083..5102
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5122..5199)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5405..5424
                     /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 cgctgctgac aaaccaagag gatttgccag aggtcttgat
      181 cctgaaagaa taattggtgc cacagacagc agtggagaat tgatgtttct catgaaatgg
      241 aaagattcag atgaggcaga cttggtgctg gcgaaagagg caaatatgaa gtgtcctcaa
      301 attgtaattg ctttttatga agagagacta acttggcatt cttgatgacg aagcttgcgg
      361 ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
      421 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
      481 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
      541 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
      601 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
      661 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
      721 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
      781 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
      841 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
      901 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
      961 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     1021 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     1081 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     1141 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     1201 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     1261 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     1321 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     1381 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     1441 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     1501 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     1561 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     1621 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     1681 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     1741 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     1801 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     1861 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     1921 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     1981 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2041 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2101 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2161 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2221 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2281 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2341 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2401 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2461 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2521 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2581 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2641 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     2701 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     2761 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     2821 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     2881 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     2941 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3001 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3061 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3121 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     3181 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     3241 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     3301 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     3361 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     3421 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     3481 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     3541 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     3601 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     3661 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     3721 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     3781 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     3841 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     3901 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     3961 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4021 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4081 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4141 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4201 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4261 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4321 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4381 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4441 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4501 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     4561 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     4621 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     4681 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     4741 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     4801 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     4861 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     4921 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     4981 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5041 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5101 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5161 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5221 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     5281 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     5341 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     5401 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     5461 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
