LOCUS       sequence-232625-        5672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5672)
  TITLE     SGC Toronto Q3-Q4 2017, Q1-Q2 2018
REFERENCE   2  (bases 1 to 5672)
  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: 6ASB (FBXL19)
FEATURES             Location/Qualifiers
     source          1..5672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             44..61
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             74..94
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             490..507
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(560..578)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      574..621
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      658..1113
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(745..764)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     955..976
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1205..2020)
                     /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(1205..2020)
                     /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     1928..1947
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2012..2031
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2142..2730
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2631..2650
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2884..2901
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2916..3055
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2981..3000)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3141..3163
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3157..3348)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4157..5239)
                     /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     5201..5220
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5240..5317)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5523..5542
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5626..5645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5626..5644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5645..5669
                     /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)."
ORIGIN
        1 ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgcatcatc atcatcatca
       61 cagcagcggc agagaaaact tgtatttcca gggcgcccgt cgccgtcgca cccgttgccg
      121 tcgctgtcgt gcatgcgttc gcacggaatg tggcgattgc catttttgtc gcgacatgaa
      181 aaaatttggc ggtccgggtc gtatgaaaca gagctgtctg ctgcgtcaat gcaccgcacc
      241 ggtgctgccg cacacggccg tttgcctgct gtgtggcgaa gccggtaaag aagataccgt
      301 cgaaggcgaa gaagaaaaat ttggtctgtc tctgatggaa tgcacgattt gtaacgaaat
      361 cgtccatccg ggttgtctga aaatgggcaa agcagaaggt gtgattaacg ctgaaatccc
      421 gaattgctgg gaatgtccgc gttgcaccca ggaaggtcgt tgatgacgaa gcttgcggcc
      481 gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
      541 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
      601 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
      661 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
      721 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
      781 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
      841 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
      901 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
      961 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     1021 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     1081 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     1141 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     1201 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     1261 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     1321 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     1381 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     1441 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     1501 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     1561 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     1621 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     1681 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     1741 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     1801 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     1861 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     1921 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     1981 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     2041 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     2101 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2161 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2221 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2281 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2341 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2401 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2461 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2521 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2581 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     2641 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2701 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2761 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     2821 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     2881 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     2941 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     3001 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     3061 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3121 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3181 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     3241 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     3301 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     3361 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     3421 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     3481 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     3541 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     3601 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     3661 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     3721 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     3781 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     3841 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     3901 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     3961 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     4021 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     4081 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     4141 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4201 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     4261 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     4321 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     4381 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     4441 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     4501 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     4561 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     4621 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     4681 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     4741 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     4801 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     4861 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     4921 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     4981 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     5041 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     5101 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     5161 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     5221 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     5281 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     5341 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     5401 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     5461 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     5521 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     5581 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaattaata cgactcacta
     5641 taggggaatt gtgagcggat aacaattccc ct
//
