LOCUS       sequence-174923-        6344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of MKK3 aa 1-347.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6344)
  TITLE     Mace Lab plasmids
REFERENCE   2  (bases 1 to 6344)
  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 MKK3 1-347
FEATURES             Location/Qualifiers
     source          1..6344
                     /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             1122..1139
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1192..1210)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1206..1253
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1290..1745
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1377..1396)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1587..1608
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1837..2652)
                     /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(1837..2652)
                     /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     2560..2579
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2644..2663
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2774..3362
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3263..3282
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3516..3533
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3548..3687
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3613..3632)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3773..3795
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3789..3980)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4789..5871)
                     /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     5833..5852
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5872..5949)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6155..6174
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6258..6277
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6258..6276
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6277..6301
                     /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             6316..6338
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atggcacatc accaccacca tcactccgcg gctcttgagg tgctctttca gggacccggt
       61 atggagtcgc ccgcctcgag ccagcccgcc agcatgcccc agtccaaagg aaaatccaag
      121 aggaagaagg atctacggat atcctgcatg tccaagccac ccgcacccaa ccccacaccc
      181 ccccggaacc tggactcccg gaccttcatc accattggag acagaaactt tgaggtggag
      241 gctgatgact tggtgaccat ctcagaactg ggccgtggag cctatggggt ggtagagaag
      301 gtgcggcacg cccagagcgg caccatcatg gccgtgaagc ggatccgggc caccgtgaac
      361 tcacaggagc agaagcggct gctcatggac ctggacatca acatgcgcac ggtcgactgc
      421 ttctacactg tcaccttcta cggggcacta ttcagagagg gagacgtgtg gatctgcatg
      481 gagctcatgg acacatcctt ggacaagttc taccggaagg tgctggataa aaacatgaca
      541 attccagagg acatccttgg ggagattgct gtgtctatcg tgcgggccct ggagcatctg
      601 cacagcaagc tgtcggtgat ccacagagat gtgaagccct ccaatgtcct tatcaacaag
      661 gagggccatg tgaagatgtg tgactttggc atcagtggct acttggtgga ctctgtggcc
      721 aagacgatgg atgccggctg caagccctac atggcccctg agaggatcaa cccagagctg
      781 aaccagaagg gctacaatgt caagtccgac gtctggagcc tgggcatcac catgattgag
      841 atggccatcc tgcggttccc ttacgagtcc tgggggaccc cgttccagca gctgaagcag
      901 gtggtggagg agccgtcccc ccagctccca gccgaccgtt tctcccccga gtttgtggac
      961 ttcactgctc agtgcctgag gaagaacccc gcagagcgta tgagctacct ggagctgatg
     1021 gagcacccct tcttcacctt gcacaaaacc aagaagacgg acattgctgc cttcgtgaag
     1081 gagatcctgg gagaagactc ataaccgggc ttctcctcga gcaccaccac caccaccact
     1141 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     1201 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     1261 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     1321 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1381 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1441 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1501 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1561 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     1621 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     1681 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     1741 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1801 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     1861 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     1921 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     1981 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     2041 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     2101 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     2161 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     2221 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     2281 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     2341 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     2401 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     2461 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     2521 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     2581 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     2641 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     2701 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2761 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2821 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2881 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     2941 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3001 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3061 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3121 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3181 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3241 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3301 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3361 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3421 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3481 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3541 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     3601 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     3661 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     3721 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3781 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     3841 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     3901 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     3961 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     4021 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     4081 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     4141 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     4201 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     4261 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     4321 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     4381 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     4441 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     4501 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     4561 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     4621 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     4681 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     4741 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     4801 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4861 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     4921 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     4981 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     5041 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     5101 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     5161 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     5221 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     5281 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     5341 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     5401 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     5461 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     5521 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     5581 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     5641 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     5701 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     5761 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     5821 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     5881 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     5941 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     6001 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     6061 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     6121 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     6181 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     6241 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     6301 ccctctagaa ataattttgt ttaactttaa gaaggagata tacc
//
