LOCUS       sequence-212286-        5944 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 5944)
  TITLE     SGC Oxford April 2018 plasmids
REFERENCE   2  (bases 1 to 5944)
  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: TRMT10C (RG9MTD1A-c113)
FEATURES             Location/Qualifiers
     source          1..5944
                     /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)."
     CDS             697..717
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             718..735
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             736..759
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             809..826
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(879..897)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      893..940
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      977..1432
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1064..1083)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1274..1295
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1524..2339)
                     /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(1524..2339)
                     /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     2247..2266
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2331..2350
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2461..3049
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2950..2969
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3203..3220
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3235..3374
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3300..3319)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3460..3482
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3476..3667)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4476..5558)
                     /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     5520..5539
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5559..5636)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5842..5861
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaacct taagaaggag atatactatg caacctttgg tttttgacat ggcttacgaa
      121 aattatatga aacgaaaaga attgcagaat actgtttccc agcttttaga aagtgaagga
      181 tggaacagaa gaaatgttga tcctttccat atttatttct gcaatctaaa aatagatggt
      241 gctttgcaca gagagttagt taaacggtat caagaaaaat gggacaaatt gcttttaaca
      301 tcaacagaaa agtctcatgt agatttattt ccaaaggaca gtattatcta tttaactgca
      361 gattctccca atgttatgac tactttcagg catgacaaag tttatgtaat tgggtctttt
      421 gttgataaga gtatgcagcc aggcacatcc ctagccaagg caaaacggct gaacctggca
      481 actgaatgcc ttccattaga taaatattta caatgggaaa ttggtaacaa aaatctcacc
      541 ttagatcaaa tgatacgtat tttgttatgt ctgaaaaaca atggtaattg gcaagaggct
      601 ctgcaattcg ttcccaagag aaaacatact ggttttctgg agatttctca gcattctcaa
      661 gagtttatca acagactaaa gaaggcaaag actgcagaga acctctactt ccaatcgcac
      721 catcatcacc accatgatta caaggatgac gacgataagt gaggatccga attcgagctc
      781 cgtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc
      841 taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
      901 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
      961 cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     1021 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     1081 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     1141 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     1201 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     1261 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     1321 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     1381 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     1441 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     1501 tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat
     1561 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     1621 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     1681 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     1741 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct
     1801 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     1861 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa
     1921 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     1981 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     2041 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     2101 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     2161 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     2221 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     2281 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata
     2341 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc
     2401 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2461 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2521 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2581 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     2641 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2701 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2761 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2821 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     2881 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     2941 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3001 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3061 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3121 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3181 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3241 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3301 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     3361 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3421 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3481 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     3541 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     3601 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     3661 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     3721 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     3781 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     3841 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     3901 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     3961 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4021 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4081 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     4141 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     4201 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     4261 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     4321 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     4381 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     4441 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4501 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4561 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     4621 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     4681 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     4741 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     4801 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     4861 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     4921 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     4981 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     5041 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     5101 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     5161 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     5221 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     5281 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     5341 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     5401 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     5461 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     5521 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     5581 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     5641 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     5701 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     5761 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     5821 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     5881 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     5941 aaat
//
