LOCUS       sequence-136219-        5692 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 5692)
  TITLE     SGC Oxford
REFERENCE   2  (bases 1 to 5692)
  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: KCTD10
FEATURES             Location/Qualifiers
     source          1..5692
                     /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             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             134..154
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             553..570
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(623..641)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      637..684
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      721..1176
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(808..827)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1018..1039
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1269..2084)
                     /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(1269..2084)
                     /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     1992..2011
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2076..2095
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2206..2794
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2695..2714
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2948..2965
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2980..3122
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3048..3067)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3208..3230
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3224..3415)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4224..5306)
                     /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     5268..5287
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5307..5384)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5590..5609
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt
      121 agatctgggt accgagaacc tgtacttcca atccatgggc acgagcccca gctccaaata
      181 cgtgaagctg aatgtgggtg gagccctcta ctatacaacc atgcagacgc tgaccaagca
      241 ggacaccatg ctgaaggcca tgttaagcgg gcgcatggaa gtgctcaccg acagtgaagg
      301 ctggatcctc attgaccgct gtgggaagca ctttggtacg atactcaact accttcgaga
      361 cggggcggtg cctttacccg agagccgccg ggagatcgag gagctgctag cagaagccaa
      421 gtactaccta gtccaaggcc tggtggaaga gtgccaggcg gccctacaaa acaaagatac
      481 ttatgagtga cagtaaaggt ggatacggat ccgaattcga gctccgtcga caagcttgcg
      541 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
      601 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
      661 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
      721 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
      781 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      841 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      901 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      961 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1021 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1081 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1141 acgcgaattt taacaaaata ttaacgttta caatttcagg tggcactttt cggggaaatg
     1201 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1261 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     1321 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     1381 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     1441 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     1501 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     1561 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     1621 cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca
     1681 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     1741 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     1801 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     1861 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     1921 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     1981 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     2041 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     2101 ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc
     2161 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2221 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2281 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2341 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2401 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2461 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2521 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2581 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2641 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2701 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2761 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2821 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2881 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     2941 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     3001 tacgcatctg tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct
     3061 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     3121 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3181 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3241 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     3301 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     3361 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     3421 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     3481 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     3541 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     3601 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     3661 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     3721 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     3781 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     3841 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     3901 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     3961 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     4021 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     4081 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     4141 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     4201 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4261 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4321 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4381 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4441 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     4501 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     4561 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     4621 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     4681 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     4741 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     4801 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     4861 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     4921 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     4981 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5041 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5101 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5161 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5221 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5281 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5341 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5401 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     5461 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     5521 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     5581 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     5641 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa at
//
