LOCUS       sequence-212338-        6228 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 6228)
  TITLE     SGC Oxford April 2018 plasmids
REFERENCE   2  (bases 1 to 6228)
  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: KLHL3 (KLHL3A-c003)
FEATURES             Location/Qualifiers
     source          1..6228
                     /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             1093..1110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1163..1181)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1177..1224
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1261..1716
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1348..1367)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1558..1579
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1808..2623)
                     /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(1808..2623)
                     /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     2531..2550
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2615..2634
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2745..3333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3234..3253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3487..3504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3519..3658
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3584..3603)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3744..3766
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3760..3951)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4760..5842)
                     /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     5804..5823
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5843..5920)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6126..6145
                     /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 atccatgagc cttcccaagg tcatgattgt
      181 ggttggcggc caggcaccca aggcaatccg cagtgtggag tgctatgatt tcgaggagga
      241 ccggtgggat cagattgctg agcttccttc cagaagatgc agagcaggtg tggtgttcat
      301 ggctggccac gtgtatgccg tgggagggtt taatggctca ctgcgggtgc ggacagtgga
      361 tgtgtatgac ggcgtgaagg accagtggac gtccattgcc agcatgcagg agcgccggag
      421 cacactgggc gcagcggtgc tcaatgactt gctctacgca gtgggaggct ttgatggcag
      481 tactggccta gcatcggtgg aagcctacag ctacaagacc aacgagtggt tctttgtggc
      541 cccgatgaac acgcggcgga gcagtgtggg tgtgggcgtt gtggagggga agctatatgc
      601 tgttgggggt tatgatggag cttcccgcca gtgtctgagc actgtggagc agtacaaccc
      661 agcgaccaat gaatggatat acgtggcgga catgagcacc cgccgcagtg gcgcaggggt
      721 tggagtgctt agcggacagc tgtacgccac aggtgggcat gatgggcctt tggtgaggaa
      781 gagcgttgag gtttacgatc ctggaacaaa tacctggaag caagtggcag acatgaacat
      841 gtgccggcgc aacgcagggg tctgtgcagt aaatgggctc ctgtatgtgg ttggagggga
      901 tgatggatcc tgcaacttgg cttcggtgga gtactacaat cctgtcactg acaaatggac
      961 gctgcttcca acgaacatga gcacggggcg gagctatgca ggtgttgccg tgattcacaa
     1021 gtccttgtga cagtaaaggt ggatacggat ccgaattcga gctccgtcga caagcttgcg
     1081 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     1141 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     1201 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     1261 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     1321 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     1381 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     1441 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     1501 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     1561 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     1621 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     1681 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     1741 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     1801 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     1861 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     1921 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     1981 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     2041 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     2101 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     2161 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     2221 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     2281 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     2341 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     2401 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     2461 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     2521 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     2581 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     2641 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     2701 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2761 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2821 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2881 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2941 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3001 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3061 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3121 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3181 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3241 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3301 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3361 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3421 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     3481 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     3541 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     3601 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     3661 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     3721 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     3781 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     3841 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     3901 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     3961 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     4021 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4081 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4141 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4201 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     4261 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     4321 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     4381 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     4441 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     4501 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     4561 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     4621 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     4681 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     4741 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     4801 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     4861 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     4921 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     4981 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     5041 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5101 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5161 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5221 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     5281 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     5341 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     5401 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     5461 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     5521 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     5581 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     5641 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     5701 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     5761 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     5821 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     5881 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     5941 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     6001 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     6061 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     6121 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     6181 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaat
//
