LOCUS       sequence-406388-        4351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ENTR Vector 4 for MultiSite Gateway assembly. Encodes eGFP under the
            control of a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4351)
  AUTHORS   Aulicino F, Pelosse M, Toelzer C, Capin J, Ilegems E, Meysami P, 
            Rollarson R, Berggren PO, Dillingham MS, Schaffitzel C, Saleem MA, 
            Welsh GI, Berger I
  TITLE     Highly efficient CRISPR-mediated large DNA docking and multiplexed 
            prime editing using a single baculovirus.
  JOURNAL   Nucleic Acids Res. 2022 Jul 22;50(13):7783-7799. doi: 
            10.1093/nar/gkac587.
  PUBMED    35801912
REFERENCE   2  (bases 1 to 4351)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2022-07-22- 22"; volume: "50"; issue: 
            "13"; pages: "7783-7799"
COMMENT     Sequence Label: pMMK ENTR 4 CMV eGFP
FEATURES             Location/Qualifiers
     source          1..4351
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    605..700
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     enhancer        712..1091
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1092..1295
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1246..1266
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1340..1349
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1346..1348,1349..1351,1352..2062)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1346..1348,1349..1351,1352..2062)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1391..1412)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1652..1671)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1999..2020
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2331..2465
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2368..2387)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2422..2441
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    2586..3174
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2639..2659)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3057..3068)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(3190..3289)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3385..3404)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3396..4205
                     /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(3463..3482)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcgaaataa tgattttatt ttgactgata gtgacctgtt cgttgcaaca aattgatgag
      661 caatgctttt ttataatgcc aactttgtat acaaaagttg gtttaaacga tgacattgat
      721 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      781 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      841 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      901 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      961 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     1021 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     1081 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
     1141 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
     1201 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
     1261 ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg
     1321 cttactggct tatgaattcg ccgccatggt gagcaagggc gaggagctgt tcaccggggt
     1381 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     1441 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     1501 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     1561 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     1621 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     1681 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1741 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     1801 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     1861 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1921 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     1981 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     2041 cggcatggac gagctgtaca agtccggact cagatctcga tagcccgggg agacccaagc
     2101 tggctagtgg atcccggtcc gaagcgcgcg gaattcaaag gcctacgtcg acgagctcac
     2161 ttgtcgcggc cgctttcgaa tctagagcct gcagtctcga caagcttgtc gagaagtact
     2221 agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     2281 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     2341 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     2401 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     2461 ggatctgatc actgcttgag cctagaagat ccggctgcta acaaagcccg aaaggaagct
     2521 gagttggctg ctgccaccgc tgagcaataa ctatcataac ccctagggta tacccatcta
     2581 attggaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     2641 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     2701 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     2761 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     2821 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     2881 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     2941 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc
     3001 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     3061 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     3121 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatccct
     3181 aggcccggga gacagctttc ttgtacaaag ttggcattat aagaaagcat tgcttatcaa
     3241 tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc tggcccgtgt
     3301 ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac
     3361 tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt
     3421 cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     3481 ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag
     3541 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     3601 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     3661 ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc caggtattag
     3721 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     3781 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     3841 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     3901 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg
     3961 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     4021 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     4081 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     4141 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     4201 tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg acttgacggg
     4261 acggcgcaag ctcatgacca aaatccctta acgtgagtta cgcgtcgttc cactgagcgt
     4321 cagaccccgt agaaaagatc aaaggatctt c
//
