LOCUS       sequence-101425-        4139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MuLE (Multiple Lentiviral Expression) Entry vector containing a CMV 
            promoter and EGFP module. Compatible with MultiSite Gateway cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4139)
  AUTHORS   Albers J, Danzer C, Rechsteiner M, Lehmann H, Brandt LP, Hejhal T, 
            Catalano A, Busenhart P, Goncalves AF, Brandt S, Bode PK, 
            Bode-Lesniewska B, Wild PJ, Frew IJ
  TITLE     A versatile modular vector system for rapid combinatorial mammalian 
            genetics.
  JOURNAL   J Clin Invest. 2015 Mar 9. pii: 79743. doi: 10.1172/JCI79743.
  PUBMED    25751063
REFERENCE   2  (bases 1 to 4139)
  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 Clin 
            Invest. 2015 Mar 9. pii: 79743. doi: 10.1172/JCI79743."
COMMENT     Sequence Label: pMuLE ENTR CMV eGFP R4-R3
FEATURES             Location/Qualifiers
     source          1..4139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    21..144
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(200..218)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(223..239)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(223..239)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(341..360)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             352..1161
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(419..438)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1308..1896
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1797..1816
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2050..2067
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2226..2253)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2345..2431)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2480..2502
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2494..2511
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2495..2511
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2561..2685)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     enhancer        2712..3091
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3092..3295
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3246..3266
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        3340..3358
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      3394..3403
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3400..3402,3403..3405,3406..4119)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3400..3402,3403..3405,3406..4116)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3445..3466)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3706..3725)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4053..4074
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 tcgagcatgc atctagagga caactttgta taataaagtt gaacgagaaa cgtaaaatga
       61 tataaatatc aatatattaa attagatttt gcataaaaaa cagactacat aatactgtaa
      121 aacacaacat atccagtcac tatgaatcaa ctacttagat ggtattagtg acctgtactg
      181 cagggcggcc gcgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg
      241 cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca
      301 tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat
      361 attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg
      421 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg
      481 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
      541 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
      601 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca
      661 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
      721 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
      781 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
      841 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
      901 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
      961 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     1021 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     1081 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     1141 atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt
     1201 acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg
     1261 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     1321 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     1381 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     1441 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     1501 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     1561 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     1621 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     1681 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     1741 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     1801 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     1861 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     1921 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     1981 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     2041 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     2101 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     2161 gcgggcagtg agcgcaacgc aattaatacg cgtaccgcta gccaggaaga gtttgtagaa
     2221 acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg
     2281 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
     2341 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     2401 agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgcgtta
     2461 acgctagcat ggatgttttc ccagtcacga cgttgtaaaa cgacggccag tcttaagctc
     2521 gggcccctac aggtcactaa taccatctaa gtagttgatt catagtgact ggatatgttg
     2581 tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata tattgatatt
     2641 tatatcattt tacgtttctc gttcaacttt tctatacaaa gttggcgatg tacgggccag
     2701 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     2761 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     2821 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     2881 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     2941 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     3001 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     3061 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
     3121 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
     3181 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
     3241 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg
     3301 gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc actataggga
     3361 gacccaagct tggtaccgag ctcggatcct aaagccacca tggtgagcaa gggcgaggag
     3421 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     3481 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     3541 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     3601 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     3661 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     3721 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     3781 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     3841 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     3901 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     3961 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     4021 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     4081 gccgggatca ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagagc
//
