LOCUS       sequence-193499-        5465 bp    DNA     circular SYN 15-APR-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 5465)
  TITLE     SGC Toronto Q4 2016, Q1 Q2 2017
REFERENCE   2  (bases 1 to 5465)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: 5W9S (CXXC5)
FEATURES             Location/Qualifiers
     source          1..5465
                     /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             330..347
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(400..418)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      414..461
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      498..953
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(585..604)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     795..816
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1045..1860)
                     /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(1045..1860)
                     /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     1768..1787
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1852..1871
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1982..2570
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2471..2490
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2724..2741
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2756..2895
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2821..2840)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2981..3003
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2997..3188)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3997..5079)
                     /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     5041..5060
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5080..5157)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5363..5382
                     /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 cagctccggc aagaagaagc ggaaacgctg cggcatgtgc
      181 gcgccctgcc ggcggcgcat caactgcgag cagtgcagca gttgtaggaa tcgaaagact
      241 ggccatcaga tttgcaaatt cagaaaatgt gaggaactca aaaagaagcc ttccgctgct
      301 tgatgacgaa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
      361 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
      421 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
      481 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
      541 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
      601 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
      661 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
      721 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
      781 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
      841 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
      901 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
      961 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1021 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     1081 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     1141 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     1201 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     1261 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     1321 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     1381 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     1441 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     1501 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     1561 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     1621 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     1681 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     1741 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     1801 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     1861 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     1921 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     1981 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2041 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     2101 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     2161 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     2221 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     2281 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     2341 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     2401 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     2461 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     2521 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     2581 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     2641 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     2701 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     2761 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     2821 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     2881 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     2941 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3001 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     3061 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     3121 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     3181 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     3241 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     3301 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     3361 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     3421 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     3481 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     3541 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     3601 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     3661 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     3721 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     3781 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     3841 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     3901 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     3961 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4021 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4081 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     4141 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     4201 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4261 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4321 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     4381 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     4441 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     4501 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     4561 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     4621 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     4681 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     4741 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     4801 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     4861 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     4921 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     4981 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     5041 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     5101 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     5161 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     5221 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     5281 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     5341 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     5401 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     5461 gaaat
//
