LOCUS       sequence-438533-        8162 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MAP2K4 (pLxIS) in mammalian cells by retroviral 
            transduction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8162)
  AUTHORS   Landau LM, Chaudhary N, Tien YC, Rogozinska M, Joshi S, Yao C, 
            Crowley J, Hullahalli K, Campbell IW, Waldor MK, Haigis M, Kagan JC
  TITLE     pLxIS-containing domains are biochemically flexible regulators of 
            interferons and metabolism.
  JOURNAL   Mol Cell. 2024 Jul 11;84(13):2436-2454.e10. doi: 
            10.1016/j.molcel.2024.05.030. Epub 2024 Jun 25.
  PUBMED    38925114
REFERENCE   2  (bases 1 to 8162)
  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"; doi: 
            "10.1016/j.molcel.2024.05.030"; journalName: "Mol Cell"; date: 
            "2024-07-11- 11"; volume: "84"; issue: "13"; pages: "2436-2454.e10"
COMMENT     Sequence Label: pMSCV-FKBP-MAP2K4
FEATURES             Location/Qualifiers
     source          1..8162
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      7..16
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             16..81
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    1785..2351
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1801..2353
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1945..1962)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2172..2191
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2365..2367,2368..2370,2371..3084)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2410..2431)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2671..2690)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3018..3039
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             3146..3660
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3822..3838
                     /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(3846..3852,3853..3870,3871..3876))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3891..3912
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4029..4046)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4200..4788)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4280..4299)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4959..5750,5751..5819))
                     /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     5582..5601
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5820..5924)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5992..6010
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6048..6070)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6170..6189
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6383..6405
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6533..6552
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             6752..7268
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    7332..7673
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             7740..8156
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     8086..8108
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     8124..8140
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
ORIGIN
        1 agatctgcca ccatggacta caaggatcac gacggtgatt acaaggacca cgacatcgac
       61 tacaaggacg acgacgacaa gggcgttcaa gtagaaacaa tatctccagg agacgggcgt
      121 acatttccta agcgtggcca aacttgcgta gtccattata ccggcatgct ggaggacggt
      181 aagaaggtcg atagctctag agaccgtaac aaacctttca aatttatgct ggggaaacag
      241 gaggttatcc gtggttggga ggaaggcgtg gcccaaatgt ccgttgggca gcgtgctaag
      301 ttaaccatct cgccagatta cgcatatggg gctacaggcc atcctgggat cattcccccg
      361 catgcgacac ttgtgtttga tgtagagctg ttgaagttgg agacgcgggg agttcaagtc
      421 gaaacgattt caccaggtga tggacgcacc tttcctaaac ggggacaaac gtgcgtcgtg
      481 cattacaccg gtatgctgga agacggcaag aaagtcgatt caagccgcga tagaaataaa
      541 cccttcaaat tcatgcttgg aaaacaggaa gtaatacgcg gatgggaaga gggtgtggcg
      601 caaatgtctg tcgggcaaag agctaaactg acaataagtc ctgattacgc gtacggtgcg
      661 actgggcacc ctggtatcat tccgccgcac gcgacacttg tcttcgacgt cgagttactt
      721 aagcttgaga cgcggggggt gcaggtagag actatttcgc ccggtgacgg aagaactttc
      781 ccaaagcggg gacagacctg tgttgtccac tacactggga tgttggagga tggtaagaaa
      841 gttgactcaa gtcgtgatcg caacaaaccc tttaagttta tgcttgggaa gcaggaagtc
      901 atcagaggtt gggaagaagg cgtagctcag atgtcagtgg gacaacgggc caaattaacc
      961 atcagtccag actacgccta cggagccacc ggtcacccag ggatcatacc acctcacgca
     1021 accttagtgt tcgatgtgga acttttaaag ttagagacgc gcggtgtaca agtggaaaca
     1081 atcagtccag gggatgggag aacattcccc aaaagaggcc agacgtgtgt cgtgcactat
     1141 acggggatgt tagaagatgg gaaaaaggtc gattcgtcac gcgaccgcaa caaaccgttt
     1201 aaattcatgt tgggaaagca ggaggtgatt cgcggctggg aggaaggtgt ggcacagatg
     1261 agcgtgggcc agagagcaaa actgactatc tctcctgatt atgcctacgg cgcgacgggc
     1321 caccctggca taattccacc ccacgctacc ctggtattcg atgtcgagct tctgaagctt
     1381 gagggtgctg gggctggggc tatggctgca ccttctccaa gtggcggtgg cggatctggt
     1441 ggaggaggtg gaacacctgg acctattgga ccacctgctt ctggccaccc ggcggtcagc
     1501 agcatgcagg gctttcagat aaacttctgt gaaaaggcac aaagtaagcg caaagcactg
     1561 aagttgaatt ttgcaaatcc acctgtcaaa tcgacagcac ggtttactct gaatcctaat
     1621 actacaggag tccagaaccc acacatagag agactgagaa cacacagcat tgagtcatca
     1681 ggaaaactga aaatttcccc tgaacaacac tgggatttca ctgcagagga ctgataagcg
     1741 gccgcgttta aacgtcgacg gtatcgataa gcttgatatc gaattccgcc ccccccccta
     1801 acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt
     1861 ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga
     1921 cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg
     1981 tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt
     2041 gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat
     2101 aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg
     2161 aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg
     2221 taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt
     2281 cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa
     2341 cacgatgata atatggccac aaccatggtg agcaagggcg aggagctgtt caccggggtg
     2401 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtctggc
     2461 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     2521 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     2581 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     2641 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     2701 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     2761 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     2821 atcatggccg acaagcagaa gaacggcatc aaggcgaact tcaagatccg ccacaacatc
     2881 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     2941 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     3001 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     3061 ggcatggacg agctgtacaa gtaatgaatt aattaagaat tcgataaaat aaaagatttt
     3121 atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg gcaagctagc
     3181 ttaagtaacg ccattttgca aggcatggaa aatacataac tgagaataga gaagttcaga
     3241 tcaaggttag gaacagagag acagcagaat atgggccaaa caggatatct gtggtaagca
     3301 gttcctgccc cggctcaggg ccaagaacag atggtcccca gatgcggtcc cgccctcagc
     3361 agtttctaga gaaccatcag atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg
     3421 ccttatttga actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc
     3481 gagctcaata aaagagccca caacccctca ctcggcgcgc cagtcctccg atagactgcg
     3541 tcgcccgggt acccgtgtat ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc
     3601 gctgttcctt gggagggtct cctctgagtg attgactacc cgtcagcggg ggtctttcat
     3661 gggtaacagt ttcttgaagt tggagaacaa cattctgagg gtaggagtcg aatattaagt
     3721 aatcctgact caattagcca ctgttttgaa tccacatact ccaatactcc tgaaatagtt
     3781 cattatggac agcgcagaaa gagctgggga gaattgtgaa attgttatcc gctcacaatt
     3841 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     3901 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3961 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4021 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4081 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4141 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4201 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4261 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4321 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4381 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4441 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4501 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4561 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4621 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4681 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4741 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4801 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4861 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4921 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4981 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5041 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5101 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5161 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5221 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5281 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5341 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5401 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5461 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5521 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5581 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5641 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5701 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5761 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5821 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5881 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5941 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     6001 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     6061 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     6121 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     6181 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     6241 aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     6301 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     6361 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc gcaaggaatg
     6421 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     6481 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     6541 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     6601 cggcgtagag gcgattagtc caatttgtta aagacaggat atcagtggtc caggctctag
     6661 ttttgactca acaatatcac cagctgaagc ctatagagta cgagccatag ataaaataaa
     6721 agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca
     6781 agctagctta agtaacgcca ttttgcaagg catggaaaat acataactga gaatagagaa
     6841 gttcagatca aggttaggaa cagagagaca gcagaatatg ggccaaacag gatatctgtg
     6901 gtaagcagtt cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtcccgc
     6961 cctcagcagt ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga
     7021 ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct
     7081 gctccccgag ctcaataaaa gagcccacaa cccctcactc ggcgcgccag tcctccgata
     7141 gactgcgtcg cccgggtacc cgtattccca ataaagcctc ttgctgtttg catccgaatc
     7201 gtggactcgc tgatccttgg gagggtctcc tcagattgat tgactgccca cctcgggggt
     7261 ctttcatttg gaggttccac cgagatttgg agacccctgc ccagggacca ccgacccccc
     7321 cgccgggagg taagctggcc agcggtcgtt tcgtgtctgt ctctgtcttt gtgcgtgttt
     7381 gtgccggcat ctaatgtttg cgcctgcgtc tgtactagtt agctaactag ctctgtatct
     7441 ggcggacccg tggtggaact gacgagttct gaacacccgg ccgcaaccct gggagacgtc
     7501 ccagggactt tgggggccgt ttttgtggcc cgacctgagg aagggagtcg atgtggaatc
     7561 cgaccccgtc aggatatgtg gttctggtag gagacgagaa cctaaaacag ttcccgcctc
     7621 cgtctgaatt tttgctttcg gtttggaacc gaagccgcgc gtcttgtctg ctgcagcgct
     7681 gcagcatcgt tctgtgttgt ctctgtctga ctgtgtttct gtatttgtct gaaaattagg
     7741 gccagactgt taccactccc ttaagtttga ccttaggtca ctggaaagat gtcgagcgga
     7801 tcgctcacaa ccagtcggta gatgtcaaga agagacgttg ggttaccttc tgctctgcag
     7861 aatggccaac ctttaacgtc ggatggccgc gagacggcac ctttaaccga gacctcatca
     7921 cccaggttaa gatcaaggtc ttttcacctg gcccgcatgg acacccagac caggtcccct
     7981 acatcgtgac ctgggaagcc ttggcttttg acccccctcc ctgggtcaag ccctttgtac
     8041 accctaagcc tccgcctcct cttcctccat ccgccccgtc tctccccctt gaacctcctc
     8101 gttcgacccc gcctcgatcc tccctttatc cagccctcac tccttctcta ggcgccggaa
     8161 tt
//
