LOCUS       sequence-194525-        5537 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 5537)
  TITLE     SGC Toronto Q2 Q3 2016
REFERENCE   2  (bases 1 to 5537)
  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: 5JJZ (CDYL2)
FEATURES             Location/Qualifiers
     source          1..5537
                     /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             94..138
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             355..372
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             402..419
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(472..490)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      486..533
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      570..1025
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(657..676)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     867..888
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1117..1932)
                     /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(1117..1932)
                     /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     1840..1859
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1924..1943
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2054..2642
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2543..2562
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2796..2813
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2828..2967
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2893..2912)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3053..3075
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3069..3260)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4069..5151)
                     /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     5113..5132
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5152..5229)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5435..5454
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg agcggcctga acgatatttt tgaagcgcag
      121 aaaattgaat ggcatgaagg cagcgctgga ggttcaggtg gggaccttta cgaggttgaa
      181 aggattgtag acaagaggaa gaacaagaaa ggaaaatggg agtatcttat ccgatggaaa
      241 ggctacggga gcaccgagga cacgtgggag ccggagcacc acctcttgca ctgtgaggag
      301 tttattgatg aattcaatgg gttgcacatg tccaaggaca agggtggaag tggtcatcac
      361 caccatcatc actgatgacg aagcttgcgg ccgcactcga gcaccaccac caccaccact
      421 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
      481 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
      541 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
      601 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
      661 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
      721 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
      781 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
      841 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
      901 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
      961 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     1021 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1081 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     1141 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     1201 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     1261 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     1321 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     1381 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     1441 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     1501 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     1561 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     1621 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     1681 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     1741 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     1801 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     1861 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     1921 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     1981 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2041 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2101 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2161 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     2221 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2281 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2341 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2401 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2461 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2521 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2581 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2641 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     2701 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     2761 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     2821 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     2881 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     2941 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     3001 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3061 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     3121 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     3181 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     3241 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     3301 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     3361 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     3421 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     3481 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     3541 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     3601 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     3661 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     3721 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     3781 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     3841 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     3901 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     3961 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     4021 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     4081 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4141 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     4201 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     4261 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     4321 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     4381 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     4441 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     4501 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     4561 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     4621 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     4681 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     4741 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     4801 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     4861 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     4921 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     4981 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     5041 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     5101 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     5161 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     5221 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     5281 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     5341 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     5401 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     5461 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     5521 tctcgatccc gcgaaat
//
