LOCUS       sequence-153004-        3433 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  nlsGFP without pA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3433)
  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 3433)
  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-nlsGFP no-pA
FEATURES             Location/Qualifiers
     source          1..3433
                     /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"
     CDS             756..776
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(822..824,825..827,828..1541)
                     /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(822..824,825..827,828..1538)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(867..888)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1128..1147)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1475..1496
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1542..1637)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1674..1693)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1675..1693)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1698..1714)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1698..1714)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1816..1835)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1827..2636
                     /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(1894..1913)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2783..3371
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3272..3291
                     /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 aaagtggaaa tggctccaaa gaagaagcgt aaggtaaatc
      781 gatcgaattc aaggcctctc gagcctctag aaggtggcgg aatggtgagc aagggcgagg
      841 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
      901 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
      961 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     1021 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1081 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1141 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1201 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1261 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     1321 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     1381 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     1441 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     1501 ccgccgggat cactctcggc atggacgagc tgtacaagta acaactttat tatacaaagt
     1561 tggcattata aaaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca
     1621 aaataaaatc attatttgga gctccatggt agcgttaacg cggccgcgat atcccctata
     1681 gtgagtcgta ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc
     1741 tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt
     1801 acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc
     1861 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     1921 ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc
     1981 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     2041 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
     2101 catggttact caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc
     2161 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
     2221 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat
     2281 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     2341 ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg
     2401 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     2461 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     2521 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     2581 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag
     2641 aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa
     2701 gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg
     2761 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2821 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2881 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     2941 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3001 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3061 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3121 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagcattgag
     3181 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3241 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3301 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3361 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3421 ttgctcacat gtt
//
