LOCUS       sequence-194704-        6995 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 6995)
  TITLE     SGC Toronto Q2 Q3 2016
REFERENCE   2  (bases 1 to 6995)
  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: 5KKP (PUS7)
FEATURES             Location/Qualifiers
     source          1..6995
                     /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"
     regulatory      1750..1759
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1860..1877
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1930..1948)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1944..1991
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2028..2483
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2115..2134)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2325..2346
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2575..3390)
                     /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(2575..3390)
                     /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     3298..3317
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3382..3401
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3512..4100
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4001..4020
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4254..4271
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4286..4425
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4351..4370)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4511..4533
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4527..4718)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5527..6609)
                     /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     6571..6590
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6610..6687)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6893..6912
                     /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 cgagagtttt gcagacatga tgaagcatgg actcactgag
      181 gctgacgtag gcatcaccaa gtttgtgagt tctcatcaag ggttctcggg aatcttaaaa
      241 gaaagatact ccgacttcgt tgttcatgaa ataggaaaag atggacggat cagccatttg
      301 aatgacttgt ccattccagt ggatgaggag gacccttcag aagacatatt tacagttttg
      361 acagctgaag aaaagcagcg attggaagag ctccagctgt tcaaaaataa ggaaaccagt
      421 gttgccattg aggttatcga ggacaccaaa gagaaaagaa ccatcatcca tcaggctatc
      481 aaatctctgt ttccaggatt agagacaaaa acagaggata gggaggggaa gaaatacatt
      541 gtagcctacc acgcagctgg gaaaaaggct ttggcaaatc caagaaaaca ttcttggcca
      601 aaatctaggg gaagttactg ccacttcgta ctatataagg aaaacaaaga caccatggat
      661 gctattaatg tactctccaa atacttaaga gtcaagccaa atatattctc ctacatggga
      721 accaaagata aaagggctat aacagttcaa gaaattgctg ttctcaaaat aactgcacaa
      781 agacttgccc acctgaataa gtgcttgatg aactttaagc tagggaattt cagctatcaa
      841 aaaaacccac tgaaattggg agagcttcaa ggaaaccact tcactgttgt tctcagaaat
      901 ataacaggaa ctgatgacca agtacagcaa gctatgaact ctctcaagga gattggattt
      961 attaactact atggaatgca aagatttgga accacagctg tccctacgta tcaggttgga
     1021 agagctatac tacaaaattc ctggacagaa gtcatggatt taatattgaa accccgctct
     1081 ggagctgaaa agggctactt ggttaaatgc agagaagaat gggcaaagac caaagaccca
     1141 actgctgccc tcagaaaact acctgtcaaa aggtgtgtgg aagggcagct gcttcgagga
     1201 ctttcaaaat atggaatgaa gaatatagtc tctgcatttg gcataatacc cagaaataat
     1261 cgcttaatgt atattcatag ctaccaaagc tatgtgtgga ataacatggt aagcaagagg
     1321 atagaagact atggactaaa acctgttcca ggggacctcg ttctcaaagg agccacagcc
     1381 acctatattg aggaagatga tgttaataat tactctatcc atgatgtggt aatgcccttg
     1441 cctggtttcg atgttatcta cccaaagcat aaaattcaag aagcctacag ggaaatgctc
     1501 acagctgaca atcttgatat tgacaacatg agacacaaaa ttcgagatta ttccttgtca
     1561 ggggcctacc gaaagatcat tattcgtcct cagaatgtta gctgggaagt cgttgcatat
     1621 gatgatccca aaattccact tttcaacaca gatgtggaca acctagaagg gaagacacca
     1681 ccagtttttg cttctgaagg caaatacagg gctctgaaaa tggatttttc tctaccccct
     1741 tctacttacg ccaccatggc cattcgagaa gtgctaaaaa tggataccag tatcaagaac
     1801 cagacgcagt tgaatacaac ctggcttcgc tgatgacgaa gcttgcggcc gcactcgagc
     1861 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     1921 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     1981 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     2041 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2101 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2161 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2221 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2281 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2341 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2401 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     2461 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     2521 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     2581 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     2641 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     2701 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     2761 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     2821 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     2881 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     2941 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     3001 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     3061 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     3121 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     3181 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     3241 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     3301 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     3361 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     3421 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     3481 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     3541 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     3601 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     3661 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     3721 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3781 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3841 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3901 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3961 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     4021 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     4081 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     4141 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     4201 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     4261 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     4321 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4381 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     4441 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     4501 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     4561 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     4621 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     4681 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     4741 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     4801 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     4861 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     4921 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     4981 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     5041 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     5101 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     5161 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     5221 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     5281 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     5341 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     5401 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     5461 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     5521 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5581 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     5641 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     5701 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     5761 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     5821 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     5881 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     5941 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     6001 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     6061 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     6121 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     6181 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     6241 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     6301 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     6361 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     6421 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     6481 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     6541 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     6601 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     6661 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     6721 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     6781 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     6841 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     6901 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     6961 cggcgtagag gatcgagatc tcgatcccgc gaaat
//
