LOCUS       sequence-161377-        3737 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  nlsGFP with pA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3737)
  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 3737)
  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 pA
FEATURES             Location/Qualifiers
     source          1..3737
                     /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"
     promoter        complement(1576..1594)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1577..1594)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1614..1631)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1620..1844
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(1846..1941)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1978..1997)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1979..1997)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2002..2018)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2002..2018)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2120..2139)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2131..2940
                     /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(2198..2217)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3087..3675
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3576..3595
                     /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 gttcatagtg 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 agcggccgct cgagcatgca
     1561 tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat cagcctcgac
     1621 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1681 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1741 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1801 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggccaact ttattataca
     1861 aagttggcat tataaaaaag cattgcttat caatttgttg caacgaacag gtcactatca
     1921 gtcaaaataa aatcattatt tggagctcca tggtagcgtt aacgcggccg cgatatcccc
     1981 tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc cgtgtctcaa
     2041 aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct
     2101 gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcgagg
     2161 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     2221 gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc gccagagttg
     2281 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     2341 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
     2401 gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt attagaagaa
     2461 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat
     2521 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg
     2581 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     2641 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca
     2701 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     2761 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     2821 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     2881 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     2941 tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg acgggacggc
     3001 gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac
     3061 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3121 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3181 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3241 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3301 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3361 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3421 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcat
     3481 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3541 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3601 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3661 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3721 ccttttgctc acatgtt
//
