LOCUS       sequence-174924-        6443 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of MKK4 aa 20-399.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6443)
  TITLE     Mace Lab plasmids
REFERENCE   2  (bases 1 to 6443)
  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: His 3C MKK4 20-399
FEATURES             Location/Qualifiers
     source          1..6443
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..24
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             34..57
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1221..1238
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1291..1309)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1305..1352
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1389..1844
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1476..1495)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1686..1707
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1936..2751)
                     /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(1936..2751)
                     /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     2659..2678
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2743..2762
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2873..3461
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3362..3381
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3615..3632
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3647..3786
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3712..3731)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3872..3894
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3888..4079)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4888..5970)
                     /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     5932..5951
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5971..6048)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6254..6273
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6357..6376
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6357..6375
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6376..6400
                     /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             6415..6437
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atggcacatc accaccacca tcactccgcg gctcttgagg tgctctttca gggacccggt
       61 acccccggcc ccgtagggtc cccggcgcca ggccacccgg ccgtcagcag catgcagggt
      121 aaacgcaaag cactgaagtt gaattttgca aatccacctt tcaaatctac agcaaggttt
      181 actctgaatc ccaatcctac aggagttcaa aacccacaca tagagagact gagaacacac
      241 agcattgagt catcaggaaa actgaagatc tcccctgaac aacactggga tttcactgca
      301 gaggacttga aagaccttgg agaaattgga cgaggagctt atggttctgt caacaaaatg
      361 gtccacaaac caagtgggca aataatggca gttaaaagaa ttcggtcaac agtggatgaa
      421 aaagaacaaa aacaacttct tatggatttg gatgtagtaa tgcggagtag tgattgccca
      481 tacattgttc agttttatgg tgcactcttc agagagggtg actgttggat ctgtatggaa
      541 ctcatgtcta cctcgtttga taagttttac aaatatgtat atagtgtatt agatgatgtt
      601 attccagaag aaattttagg caaaatcact ttagcaactg tgaaagcact aaaccactta
      661 aaagaaaact tgaaaattat tcacagagat atcaaacctt ccaatattct tctggacaga
      721 agtggaaata ttaagctctg tgacttcggc atcagtggac agcttgtgga ctctattgcc
      781 aagacaagag atgctggctg taggccatac atggcacctg aaagaataga cccaagcgca
      841 tcacgacaag gatatgatgt ccgctctgat gtctggagtt tggggatcac attgtatgag
      901 ttggccacag gccgatttcc ttatccaaag tggaatagtg tatttgatca actaacacaa
      961 gtcgtgaaag gagatcctcc gcagctgagt aattctgagg aaagggaatt ctccccgagt
     1021 ttcatcaact ttgtcaactt gtgccttacg aaggatgaat ccaaaaggcc aaagtataaa
     1081 gagcttctga aacatccctt tattttgatg tatgaagaac gtgccgttga ggtcgcatgc
     1141 tatgtttgta aaatcctgga tcaaatgcca gctactccca gctctcccat gtatgtcgat
     1201 taaccgggct tctcctcgag caccaccacc accaccactg agatccggct gctaacaaag
     1261 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     1321 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     1381 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1441 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     1501 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1561 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     1621 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1681 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     1741 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     1801 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     1861 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1921 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     1981 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     2041 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     2101 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     2161 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     2221 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     2281 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     2341 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     2401 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     2461 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     2521 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     2581 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     2641 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     2701 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     2761 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     2821 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2881 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2941 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3001 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3061 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3121 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3181 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3241 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3301 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3361 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3421 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3481 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3541 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3601 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3661 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     3721 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3781 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3841 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3901 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3961 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     4021 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     4081 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     4141 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     4201 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     4261 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     4321 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     4381 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4441 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4501 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     4561 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     4621 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     4681 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     4741 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     4801 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     4861 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4921 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4981 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     5041 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     5101 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     5161 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     5221 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     5281 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     5341 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     5401 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     5461 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5521 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5581 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5641 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     5701 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     5761 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     5821 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     5881 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     5941 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     6001 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     6061 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     6121 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     6181 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     6241 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     6301 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
     6361 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
     6421 taactttaag aaggagatat acc
//
