LOCUS       sequence-163170-        4629 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMVmin-promoted nlsGFP with pA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4629)
  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 4629)
  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:nlsGFP pA
FEATURES             Location/Qualifiers
     source          1..4629
                     /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"
     primer_bind     complement(773..792)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        964..1343
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1344..1547
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1498..1518
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        1592..1610
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1648..1668
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1714..1716,1717..1719,1720..2433)
                     /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(1714..1716,1717..1719,1720..2430)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1759..1780)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2020..2039)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2367..2388
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2468..2486)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2469..2486)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2506..2523)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2512..2736
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    complement(2738..2833)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(2871..2889)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2894..2910)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2894..2910)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3012..3031)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3023..3832
                     /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(3090..3109)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3979..4567
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4468..4487
                     /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 aaagtggccg atcccctatg gtgcactctc agtacaatct
      781 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
      841 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
      901 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
      961 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     1021 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     1081 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     1141 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     1201 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     1261 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     1321 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     1381 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     1441 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     1501 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     1561 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     1621 cttggtaccg agctcggatc catggctcca aagaagaagc gtaaggtaaa tcgatcgaat
     1681 tcaaggcctc tcgagcctct agaaggtggc ggaatggtga gcaagggcga ggagctgttc
     1741 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1801 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1861 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     1921 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1981 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     2041 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2101 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     2161 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     2221 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     2281 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     2341 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2401 atcactctcg gcatggacga gctgtacaag taagcggccg ctcgagcatg catctagagg
     2461 gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg actgtgcctt
     2521 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttcctttacc ctggaaggtg
     2581 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     2641 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     2701 atagcaggca tgctggggat gcggtgggct ctatggccaa ctttattata caaagttggc
     2761 attataaaaa agcattgctt atcaatttgt tgcaacgaac aggtcactat cagtcaaaat
     2821 aaaatcatta tttggagctc catggtagcg ttaacgcggc cgcgatatcc cctatagtga
     2881 gtcgtattac atggtcatag ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg
     2941 atgttacatt gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat
     3001 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt
     3061 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     3121 atcaggtgcg acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa
     3181 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     3241 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     3301 gttactcacc actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga
     3361 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     3421 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg
     3481 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     3541 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     3601 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     3661 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     3721 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     3781 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt
     3841 ggttaattgg ttgtaacact ggcagagcat tacgctgact tgacgggacg gcgcaagctc
     3901 atgaccaaaa tcccttaacg tgagttacgc gtcgttccac tgagcgtcag accccgtaga
     3961 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4021 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4081 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     4141 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4201 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4261 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4321 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc attgagaaag
     4381 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4441 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4501 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4561 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4621 tcacatgtt
//
