LOCUS       sequence-212284-        5646 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 5646)
  TITLE     SGC Oxford April 2018 plasmids
REFERENCE   2  (bases 1 to 5646)
  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: WNK2 (WNK2A-c003)
FEATURES             Location/Qualifiers
     source          1..5646
                     /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             511..528
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(581..599)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      595..642
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      679..1134
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(766..785)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     976..997
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1226..2041)
                     /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(1226..2041)
                     /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     1949..1968
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2033..2052
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2163..2751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2652..2671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2905..2922
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2937..3076
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3002..3021)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3162..3184
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3178..3369)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4178..5260)
                     /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     5222..5241
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5261..5338)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5544..5563
                     /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 atccatggca gaggacacag gcgtgagggt
      181 ggagctcgcg gaggaggacc acggcaggaa gtccaccatc gccctgaggc tctgggtgga
      241 agaccccaag aaactgaagg gaaagcccaa ggacaatgga gccatagagt tcaccttcga
      301 cctggagaag gagacgccgg atgaggtggc ccaagagatg attgagtctg gattcttcca
      361 cgagagtgac gtcaagatcg tggccaagtc catccgtgac cgcgtggcct tgatccagtg
      421 gcggcgggag aggatctggc ccgcgtgaca gtaaaggtgg atacggatcc gaattcgagc
      481 tccgtcgaca agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct
      541 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
      601 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
      661 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      721 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
      781 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      841 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
      901 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
      961 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     1021 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     1081 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     1141 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1201 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     1261 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     1321 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc
     1381 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     1441 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     1501 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     1561 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     1621 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     1681 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     1741 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     1801 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     1861 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     1921 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     1981 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     2041 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     2101 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2161 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2221 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2281 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     2341 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2401 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2461 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2521 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2581 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2641 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2701 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     2761 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     2821 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     2881 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     2941 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     3001 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     3061 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     3121 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     3181 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     3241 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     3301 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     3361 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     3421 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     3481 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     3541 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     3601 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     3661 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     3721 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     3781 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     3841 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     3901 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     3961 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     4021 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     4081 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     4141 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4201 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4261 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     4321 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     4381 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     4441 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     4501 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     4561 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     4621 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     4681 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     4741 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     4801 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     4861 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     4921 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     4981 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     5041 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     5101 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     5161 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     5221 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     5281 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     5341 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     5401 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     5461 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     5521 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     5581 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg
     5641 cgaaat
//
