LOCUS       sequence-193413-        6262 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 6262)
  TITLE     SGC Oxford 2017
REFERENCE   2  (bases 1 to 6262)
  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: KLHL40 (KBTBD5A-c006)
FEATURES             Location/Qualifiers
     source          1..6262
                     /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             1015..1035
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1036..1053
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1054..1077
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1127..1144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1197..1215)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1211..1258
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1295..1750
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1382..1401)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1592..1613
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1842..2657)
                     /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(1842..2657)
                     /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     2565..2584
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2649..2668
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2779..3367
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3268..3287
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3521..3538
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3553..3692
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3618..3637)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3778..3800
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3794..3985)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4794..5876)
                     /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     5838..5857
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5877..5954)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6160..6179
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaacct taagaaggag atatactatg ttcctgcagg atctcatctt catgatcagt
      121 gaggagggcg ctgtggccta cgatccagca gccaacgagt gctactgtgc ttccctctcc
      181 agccaggtcc ccaagaacca cgtcagcctg gttaccaagg agaaccaggt cttcgtggct
      241 ggaggcctct tctacaacga agacaacaaa gaggacccca tgagcgcata cttcctgcag
      301 tttgaccatc tggactcaga gtggctgggg atgccaccgc tgccctcgcc ccgctgcctc
      361 tttggcctgg gagaagctct caactccatc tacgtggtcg gtggcagaga gatcaaggac
      421 ggcgagcgct gcctggactc ggtcatgtgc tacgacaggc tgtcattcaa atggggtgaa
      481 tcggacccgc tgccttacgt ggtgtatggc cacacagtgc tctcccacat ggaccttgtc
      541 tacgtaattg gcggcaaagg cagtgacagg aagtgcctga acaagatgtg cgtctatgac
      601 cccaagaagt ttgagtggaa ggagctggca cccatgcaga ccgcccgctc actctttggg
      661 gccactgtcc atgatggccg cattatcgtg gcagctgggg tcaccgacac agggctgacc
      721 agttctgccg aagtgtacag catcacagac aacaagtggg cacccttcga ggccttccca
      781 caggagcgta gctcactcag cctggtcagc ctggtgggta ccctctatgc cattggtggc
      841 tttgccacac tggagacgga gtctggagag ctggttccca cagagctcaa tgacatctgg
      901 aggtataacg aggaggagaa gaaatgggag ggtgtcctgc gggagatcgc ctatgcagca
      961 ggtgccacct tcctaccagt gcggctcaat gtgctgcgcc tgactaagat ggcagagaac
     1021 ctctacttcc aatcgcacca tcatcaccac catgattaca aggatgacga cgataagtga
     1081 ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca
     1141 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1201 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1261 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1321 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1381 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1441 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1501 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1561 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1621 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1681 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1741 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1801 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     1861 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     1921 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     1981 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2041 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2101 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2161 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     2221 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2281 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2341 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2401 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2461 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2521 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2581 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2641 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     2701 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2761 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2821 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2881 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     2941 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3001 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3061 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3121 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3181 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3241 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3301 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3361 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3421 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3481 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3541 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3601 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3661 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3721 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3781 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     3841 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     3901 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     3961 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     4021 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     4081 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     4141 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     4201 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     4261 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     4321 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     4381 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     4441 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4501 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4561 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     4621 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     4681 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4741 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     4801 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4861 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     4921 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     4981 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     5041 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     5101 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     5161 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     5221 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     5281 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     5341 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     5401 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     5461 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5521 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5581 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5641 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5701 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     5761 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     5821 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     5881 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     5941 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     6001 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     6061 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     6121 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     6181 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
     6241 cgagatctcg atcccgcgaa at
//
