LOCUS       sequence-213451-        6854 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expression of a dominant negative MAP Kinase Kinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6854)
  AUTHORS   Seger R, Seger D, Reszka AA, Munar ES, Eldar-Finkelman H, 
            Dobrowolska G, Jensen AM, Campbell JS, Fischer EH, Krebs EG
  TITLE     Overexpression of mitogen-activated protein kinase kinase (MAPKK) 
            and its mutants in NIH 3T3 cells. Evidence that MAPKK involvement in
            cellular proliferation is regulated by phosphorylation of serine 
            residues in its kinase subdomains VII and VIII.
  JOURNAL   J Biol Chem. 1994 Oct 14;269(41):25699-709.
  PUBMED    7929275
REFERENCE   2  (bases 1 to 6854)
  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"; journalName: "J Biol 
            Chem."; date: "1994-10-14"; volume: "269(41)"; pages: "25699-709"
COMMENT     Sequence Label: pcDNA3-MKK1K97A
FEATURES             Location/Qualifiers
     source          1..6854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      916..925
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2407..2425)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2408..2425)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2445..2462)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2451..2675
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2721..3149
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2808..2827)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3018..3039
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3158..3178)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3163..3492
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3343..3478
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3405..3424
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3559..4353
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3613..3632)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4223..4242
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4527..4648
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4564..4583)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4618..4637
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4697..4713)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4697..4713)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4710..4732)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4721..4737
                     /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)."
     promoter        complement(join(4745..4751,4752..4769,4770..4775))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4790..4811
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4928..4945)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5099..5687)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5179..5198)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5858..6649,6650..6718))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6481..6500
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6719..6823)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccgccgccac catggcaccc aagaagaagc cgacgcccat ccagctgaac
      961 ccggcccccg acggctctgc agttaacggg accagctctg cggagaccaa cttggaggcc
     1021 ttgcagaaga agctggagga gctagagctt gatgagcagc agcgaaagcg ccttgaggcc
     1081 tttcttaccc agaagcagaa ggtgggagaa ctgaaggatg acgactttga gaagatcagt
     1141 gagctggggg ctggcaatgg cggtgtggtg ttcaaggtct cccacaagcc ttctggcctg
     1201 gtcatggcca gagcgctaat tcatctggag atcaaacccg caatccggaa ccagatcata
     1261 agggagctgc aggttctgca tgagtgcaac tctccgtaca tcgtgggctt ctatggtgcg
     1321 ttctacagcg atggcgagat cagtatctgc atggagcaca tggatggagg ttctctggat
     1381 caagtcctga agaaagctgg aagaattcct gaacaaattt taggaaaagt tagcattgct
     1441 gtaataaaag gcctgacata tctgagggag aagcacaaga tcatgcacag agatgtcaag
     1501 ccctccaaca tcctagtcaa ctcccgtggg gagatcaagc tctgtgactt tggggtcagc
     1561 gggcagctca tcgactccat ggccaactcc ttcgtgggca caaggtccta catgtcgcca
     1621 gaaagactcc aggggactca ttactctgtg cagtcagaca tctggagcat gggactgtct
     1681 ctggtagaga tggcggttgg gaggtatccc atccctcctc cagatgccaa ggagctggag
     1741 ctgatgtttg ggtgccaggt ggaaggagat gcggctgaga ccccacccag gccaaggacc
     1801 cccgggaggc cccttagctc atacggaatg gacagccgac ctcccatggc aatttttgag
     1861 ttgttggatt acatagtcaa cgagcctcct ccaaaactgc ccagtggagt gttcagtctg
     1921 gaatttcaag attttgtgaa taaatgctta ataaaaaacc ccgcagagag agcagatttg
     1981 aagcaactca tggttcatgc ttttatcaag agatctgatg ctgaggaagt ggattttgca
     2041 ggttggctct gctccaccat cggccttaac cagcccagca caccaaccca tgctgctggc
     2101 gtctaagtgt ttgggaagca acaaagagcg agtcccctgc ccggtggttt gccatgtcgc
     2161 ttttgggcct ccttcccatg cctgtctctg ttcagatgtg catttcacct gtgacaaagg
     2221 atgaagaaca cagcatgtgc caagattcta ctcttgtcat ttttaatatt actgtcttta
     2281 ttcttattac tattattgtt cccctaagtg gattggcttt gtgcttgggg ctatttgtgt
     2341 gtatccgaat tctgcagata tccatcacac tggcggccgc tcgagcatgc atctagaggg
     2401 ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga ctgtgccttc
     2461 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     2521 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     2581 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2641 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     2701 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     2761 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     2821 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     2881 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     2941 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3001 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3061 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3121 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3181 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3241 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3301 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3361 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3421 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3481 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     3541 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     3601 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     3661 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     3721 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     3781 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     3841 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     3901 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     3961 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4021 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4081 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4141 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4201 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4261 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4321 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4381 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     4441 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4501 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4561 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4621 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4681 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4741 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4801 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     4861 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4921 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4981 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5041 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5101 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5161 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5221 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5281 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5341 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5401 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5461 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5521 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5581 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5641 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5701 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5761 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5821 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5881 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5941 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6001 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6061 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6121 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6181 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6241 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6301 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6361 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6421 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6481 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6541 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6601 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6661 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6721 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6781 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6841 tgccacctga cgtc
//
