LOCUS       sequence-194706-        6245 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 6245)
  TITLE     SGC Toronto Q2 Q3 2016
REFERENCE   2  (bases 1 to 6245)
  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: 5JJX (SART3)
FEATURES             Location/Qualifiers
     source          1..6245
                     /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             1110..1127
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1180..1198)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1194..1241
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1278..1733
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1365..1384)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1575..1596
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1825..2640)
                     /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(1825..2640)
                     /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     2548..2567
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2632..2651
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2762..3350
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3251..3270
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3504..3521
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3536..3675
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3601..3620)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3761..3783
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3777..3968)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4777..5859)
                     /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     5821..5840
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5860..5937)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6143..6162
                     /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 cggggagtac gagtgggaat atgacgaaga ggaggagaaa
      181 aaccagctgg agattgagag actggaggag cagttgtcta tcaacgtcta tgactacaac
      241 tgccatgtgg acttgatcag actgctcagg ctggaagggg agcttaccaa ggtgaggatg
      301 gcccgccaga agatgagtga aatctttccc ttgactgaag agctctggct ggagtggctg
      361 catgacgaga tcagcatggc ccaggatggc ctggacagag agcacgtgta tgacctcttt
      421 gagaaagccg tgaaggatta catttgtcct aacatttggc tagagtatgg ccagtactca
      481 gttggtggga ttggtcagaa aggtggcctt gagaaagttc gctccgtgtt tgaaagggct
      541 ctctcgtctg ttggtttaca tatgaccaaa ggactcgccc tctgggaggc ttaccgagag
      601 tttgaaagtg cgattgtgga agctgctcgg cttgagaaag tccacagtct tttccggcga
      661 cagttggcga tcccactcta tgatatggag gccacatttg cagagtatga agaatggtca
      721 gaagacccaa taccagagtc agtaattcag aactataaca aagcactaca gcagctggag
      781 aaatataaac cctatgaaga agcactgttg caggcagagg caccaaggct ggcagaatat
      841 caagcatata tcgattttga gatgaaaatt ggcgatcctg ctcgcattca gttgatcttt
      901 gagcgcgccc tggtcgagaa ctgccttgtc ccagacttat ggatccgtta cagtcagtac
      961 ctagatcgac aactgaaagt aaaggatttg gttttatctg tacataaccg cgctattaga
     1021 aactgcccct ggacagttgc cttatggagt cggtacctct tggccatgga gagacatgga
     1081 tgatgacgaa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
     1141 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     1201 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1261 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1321 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1381 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     1441 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1501 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     1561 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     1621 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1681 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     1741 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1801 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     1861 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     1921 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     1981 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     2041 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     2101 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     2161 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     2221 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     2281 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     2341 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     2401 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     2461 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     2521 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     2581 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     2641 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     2701 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2761 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2821 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     2881 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     2941 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3001 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3061 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3121 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3181 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3241 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3301 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3361 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3421 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3481 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     3541 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     3601 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     3661 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3721 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3781 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     3841 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     3901 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     3961 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     4021 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     4081 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     4141 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     4201 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     4261 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     4321 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     4381 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     4441 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     4501 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     4561 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     4621 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     4681 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     4741 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4801 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4861 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     4921 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     4981 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     5041 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     5101 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     5161 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     5221 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     5281 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     5341 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     5401 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     5461 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     5521 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     5581 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     5641 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     5701 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     5761 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     5821 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     5881 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     5941 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     6001 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     6061 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     6121 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     6181 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     6241 gaaat
//
