LOCUS       sequence-212337-        6204 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 6204)
  TITLE     SGC Oxford April 2018 plasmids
REFERENCE   2  (bases 1 to 6204)
  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: WNK3 (WNK3B-c016)
FEATURES             Location/Qualifiers
     source          1..6204
                     /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             1069..1086
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1139..1157)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1153..1200
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1237..1692
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1324..1343)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1534..1555
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1784..2599)
                     /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(1784..2599)
                     /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     2507..2526
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2591..2610
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2721..3309
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3210..3229
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3463..3480
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3495..3634
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3560..3579)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3720..3742
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3736..3927)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4736..5818)
                     /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     5780..5799
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5819..5896)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6102..6121
                     /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 gaaatgaagg ctgtagctac
      181 ttctcctagt ggcagattcc tgaaatttga catagaacta ggaagaggag catttaaaac
      241 agtatataaa ggactggaca ctgaaacatg ggttgaggtt gcttggtgtg agctgcagga
      301 ccgaaagtta accaaagctg agcagcaaag attcaaggaa gaagcagaga tgttgaaggg
      361 tctccagcac cccaatatag ttcgatttta tgattcctgg gaatctatat taaaaggaaa
      421 gaaatgtatt gtattagtga ctgaactaat gacatctggg accttaaaga cgtacttaaa
      481 acgatttaaa gtcatgaaac caaaggtctt aaggagctgg tgcaggcaaa ttttaaaggg
      541 gttgcagttc ttgcacacta ggactcctcc tattattcac cgggatctga agtgtgacaa
      601 tattttcatc acgggaccca ctggatctgt gaagattggt gatctaggat tagccacctt
      661 aatgcgtacc tcatttgcta agagtgtcat tggaactcct gagtttatgg ctccagagat
      721 gtatgaagaa cactatgatg aatccgtaga tgtttatgct tttggaatgt gtatgctgga
      781 aatggccaca tcggagtatc cttattctga gtgtcagaat gcagctcaaa tataccggaa
      841 agtaactagt ggcataaaac cagccagctt caataaagtc actgatcctg aagtcaaaga
      901 aatcattgaa ggatgtattc gtcaaaacaa atctgaaagg ttgtctatca gggacctatt
      961 aaaccatgca ttttttgctg aggatacagg actgagggtg gagtgacagt aaaggtggat
     1021 acggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac
     1081 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1141 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1201 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     1261 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1321 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1381 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1441 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1501 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1561 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1621 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1681 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1741 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     1801 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     1861 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     1921 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     1981 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     2041 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     2101 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     2161 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     2221 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     2281 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     2341 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     2401 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     2461 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     2521 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     2581 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     2641 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2701 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2761 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2821 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2881 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2941 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3001 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3061 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3121 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3181 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3241 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3301 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3361 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3421 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3481 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3541 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     3601 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     3661 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3721 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     3781 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     3841 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     3901 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     3961 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     4021 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     4081 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4141 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4201 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     4261 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4321 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4381 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     4441 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     4501 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     4561 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     4621 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     4681 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     4741 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4801 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     4861 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     4921 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     4981 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     5041 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     5101 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     5161 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     5221 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     5281 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     5341 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     5401 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     5461 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     5521 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     5581 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     5641 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     5701 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     5761 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     5821 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     5881 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     5941 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     6001 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     6061 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     6121 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     6181 atcgagatct cgatcccgcg aaat
//
