LOCUS       sequence-152897-        4166 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMVmin-promoted GFP without pA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4166)
  AUTHORS   Fowler DK, Stewart S, Seredick S, Eisen JS, Stankunas K, Washbourne 
            P
  TITLE     A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate 
            Expression Constructs with Diverse Research Applications.
  JOURNAL   PLoS One. 2016 Aug 8;11(8):e0159277. doi: 
            10.1371/journal.pone.0159277. eCollection 2016.
  PUBMED    27500400
REFERENCE   2  (bases 1 to 4166)
  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: "PLoS One.";
            date: "2016-08-8"; volume: "11(8):e0159277. doi"; pages: " 
            10.1371/journal.pone.0159277. eCollection 2016"
COMMENT     Sequence Label: p3E-CMVmin:GFP no-pA
FEATURES             Location/Qualifiers
     source          1..4166
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(624..747)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     enhancer        889..1268
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1269..1472
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1423..1443
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        1517..1535
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1549..1558
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1555..1557,1558..1560,1561..2274)
                     /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(1555..1557,1558..1560,1561..2271)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1600..1621)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1861..1880)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2208..2229
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2275..2370)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2408..2426)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2431..2447)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2431..2447)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2549..2568)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2560..3369
                     /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(2627..2646)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3516..4104
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4005..4024
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac
      601 taataccatc taagtagttg attcatagtg actgcatatg ttgtgtttta cagtattatg
      661 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
      721 ctcgttcaac tttcttgtac aaagtgggtg tgttggaggt cgctgagtag tgcgcgagca
      781 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
      841 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
      901 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
      961 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     1021 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     1081 gtcaatgggt ggactattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     1141 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     1201 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     1261 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     1321 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     1381 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     1441 gtgtacggtg ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt
     1501 actggcttat cgaaattaat acgactcact atagggagac ccaagcttgc caccatggtg
     1561 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1621 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1681 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1741 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     1801 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1861 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1921 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1981 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     2041 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     2101 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     2161 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     2221 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaacaactt
     2281 tattatacaa agttggcatt ataaaaaagc attgcttatc aatttgttgc aacgaacagg
     2341 tcactatcag tcaaaataaa atcattattt ggagctccat ggtagcgtta acgcggccgc
     2401 gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc
     2461 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     2521 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     2581 acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct
     2641 cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg
     2701 ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg
     2761 gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt
     2821 actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta
     2881 ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc
     2941 cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc
     3001 gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag
     3061 cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca
     3121 ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg
     3181 aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt
     3241 gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa
     3301 aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag
     3361 tttttctaat cagaattggt taattggttg taacactggc agagcattac gctgacttga
     3421 cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga
     3481 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3541 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3601 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3661 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3721 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3781 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3841 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3901 cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3961 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4021 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4081 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4141 ttttgctggc cttttgctca catgtt
//
