LOCUS       sequence-101464-        3356 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MuLE (Multiple Lentiviral Expression) Entry vector containing EGFP. 
            Compatible with MultiSite Gateway cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3356)
  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 3356)
  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 eGFP L5-L2
FEATURES             Location/Qualifiers
     source          1..3356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(21..120)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(150..169)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(151..169)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(174..190)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(174..190)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(292..311)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             303..1112
                     /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(370..389)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1259..1847
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1748..1767
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2001..2018
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2177..2204)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2296..2382)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2431..2453
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2445..2462
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2446..2462
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2479..2574
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      2611..2620
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2617..2619,2620..2622,2623..3336)
                     /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(2617..2619,2620..2622,2623..3333)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2662..2683)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2923..2942)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3270..3291
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 tcgagcatgc atctagaggt acccagcttt cttgtacaaa gttggcatta taagaaagca
       61 ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa tcattatttg
      121 ccatccagct gcagggcggc cgcgatatcc cctatagtga gtcgtattac atggtcatag
      181 ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata
      241 aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg
      301 ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg
      361 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc
      421 gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg
      481 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc
      541 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc
      601 ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg
      661 atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta
      721 acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg
      781 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa
      841 tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg
      901 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa
      961 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt
     1021 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc
     1081 agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact
     1141 ggcagagcat tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg
     1201 tgagttacgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     1261 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     1321 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     1381 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     1441 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     1501 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     1561 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     1621 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     1681 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     1741 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     1801 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     1861 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     1921 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     1981 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     2041 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     2101 ccgactggaa agcgggcagt gagcgcaacg caattaatac gcgtaccgct agccaggaag
     2161 agtttgtaga aacgcaaaaa ggccatccgt caggatggcc ttctgcttag tttgatgcct
     2221 ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca caacgttcaa
     2281 atccgctccc ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa
     2341 acgaaaggcc cagtcttccg actgagcctt tcgttttatt tgatgcctgg cagttcccta
     2401 ctctcgcgtt aacgctagca tggatgtttt cccagtcacg acgttgtaaa acgacggcca
     2461 gtcttaagct cgggccccaa ataatgattt tattttgact gatagtgacc tgttcgttgc
     2521 aacaaattga tgagcaatgc ttttttataa tgccaacttt gtatacaaaa gttgggagac
     2581 ccaagcttgg taccgagctc ggatcctaaa gccaccatgg tgagcaaggg cgaggagctg
     2641 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     2701 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     2761 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     2821 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2881 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2941 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     3001 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     3061 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     3121 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     3181 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     3241 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     3301 gggatcactc tcggcatgga cgagctgtac aagtaaagcg gccgcgactc tagagc
//
