LOCUS       sequence-415188-        5001 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector driving nls-EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5001)
  AUTHORS   Ariotti N, Hall TE, Rae J, Ferguson C, McMahon KA, Martel N, Webb 
            RE, Webb RI, Teasdale RD, Parton RG
  TITLE     Modular Detection of GFP-Labeled Proteins for Rapid Screening by 
            Electron Microscopy in Cells and Organisms.
  JOURNAL   Dev Cell. 2015 Nov 23;35(4):513-25. doi: 
            10.1016/j.devcel.2015.10.016. Epub 2015 Nov 12.
  PUBMED    26585296
REFERENCE   2  (bases 1 to 5001)
  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: 
            "10.1016/j.devcel.2015.10.016"; journalName: "Dev Cell"; date: 
            "2015-11-23- 23"; volume: "35"; issue: "4"; pages: "513-25"
COMMENT     Sequence Label: nls-EGFP
FEATURES             Location/Qualifiers
     source          1..5001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    108..132
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      136..145
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             148..168
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     protein_bind    complement(173..197)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      196..205
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(202..204,205..207,208..918)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(202..204,205..207,208..918)
                     /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(247..268)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(508..527)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     855..876
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(972..990)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    995..1129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(1192..1212)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    1252..1386
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1499..1519)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1499..1517)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1538..1554)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1538..1554)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1551..1573)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1562..1578
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1586..1592,1593..1610,1611..1616))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1631..1652
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1769..1786)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1940..2528)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2020..2039)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2699..3490,3491..3559))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3322..3341
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3560..3664)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3690..4145)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3827..3848)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4039..4058
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattca aggatcaaca agtttgtaca
      121 aaaaagcagg ctgccgccac catggctcca aagaagaagc gtaaggtacg gtacccagct
      181 ttcttgtaca aagtggccac catggtgagc aagggcgagg agctgttcac cggggtggtg
      241 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      301 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
      361 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
      421 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
      481 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
      541 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
      601 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
      661 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
      721 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
      781 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
      841 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
      901 atggacgagc tgtacaaggg cggtggaaga tctgggaatt caaggcctct cgagcctcta
      961 gattctgcag ccctatagtg agtcgtatta cgtagatcca gacatgataa gatacattga
     1021 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     1081 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     1141 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt ccaactttat
     1201 tatacatagt tgcactcgag cctctagaac tatagtgagt cgtattacgt agatccagac
     1261 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     1321 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     1381 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     1441 gttttttaat tcgcggccgc ggcgccaatg cattgggccc ggtacccagc ttttgttccc
     1501 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     1561 attgttatcc gctcacaatt ccacacaaca tacgagccgg gagcataaag tgtaaagcct
     1621 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1681 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1741 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     1801 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     1861 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     1921 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     1981 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2041 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2101 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2161 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     2221 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     2281 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2341 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2401 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2461 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2521 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2581 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2641 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2701 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2761 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2821 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2881 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     2941 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3001 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3061 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3121 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3181 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3241 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3301 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3361 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3421 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3481 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     3541 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     3601 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3661 cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt
     3721 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     3781 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     3841 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     3901 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     3961 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     4021 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     4081 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     4141 cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     4201 ttcgctatta cgccagtcga ccgccaattc aatatggcgt atatggactc atgccaattc
     4261 aatatggtgg atctggacct gtgccaattc aatatggcgt atatggactc gtgccaattc
     4321 aatatggtgg atctggaccc cagccaattc aatatggccc tatgccaata tggctattgg
     4381 ccaggttcaa tactatgtat tggccctatg ccatatagta ttccatatat gggttttcct
     4441 attgacgtag atagcccctc ccaatgggcg gtcccatata ccatatatgg ggcttcctaa
     4501 taccgcccat agccactccc ccattgacgt caatggtctc tatatatggt ctttcctatt
     4561 gacgtcatat gggcggtcct attgacgtat atggcgcctc ccccattgac gtcaattacg
     4621 gtaaatggcc cgcctggctc aatgcccatt gacgtcaata ggaccaccca ccattgacgt
     4681 caatgggatg gctcattgcc cattcatatc cgttctcacg ccccctattg acgtcaatga
     4741 cggtaaatgg cccacttggc agtacatcaa tatctattaa tagtaacttg gcaagtacat
     4801 tactattgga agtacgccag ggtacattgg cagtactccc attgacgtca atggcggtaa
     4861 atggcccgcg atggctgcca agtacatccc cattgacgtc aatggggagg ggcaatgacg
     4921 caaatgggcg ttccattgac gtaaatgggc ggtaggcgtg cctaatggga ggtctatata
     4981 agcaatgctc gtttagggaa c
//
