LOCUS       sequence-72945-e        4700 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector: Position 4', Flag tagged nls-mCherry with 
            start and stop codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4700)
  AUTHORS   Kirchmaier S, Lust K, Wittbrodt J
  TITLE     Golden GATEway cloning--a combinatorial approach to generate fusion 
            and recombination constructs.
  JOURNAL   PLoS One. 2013 Oct 7;8(10):e76117. doi: 
            10.1371/journal.pone.0076117.
  PUBMED    24116091
REFERENCE   2  (bases 1 to 4700)
  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: "2013-10-7"; volume: "8(10):e76117. doi"; pages: " 
            10.1371/journal.pone.0076117"
COMMENT     Sequence Label: pGGEV_4'_+nls-mCherry-Flag-_+1_BK+
FEATURES             Location/Qualifiers
     source          1..4700
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     350..369
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        971..989
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     971..988
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      1009..1018
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1021..1041
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1042..1749
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGREFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1187..1205)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1455..1474
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1649..1672
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             1750..1773
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     intron          1848..2420
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     polyA_signal    2493..2627
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2517..2536)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2571..2590
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2779..2796)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2950..3538)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(3709..4500,4501..4569))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAISMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4332..4351
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4570..4674)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
      121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctacgca
      481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagggc gcctccccca
      541 ttgacgtcaa ttacggtaaa tggcccgcct ggctcaatgc ccattgacgt caataggacc
      601 acccaccatt gacgtcaatg ggatggctca ttgcccattc atatccgttc tcacgccccc
      661 tattgacgtc aatgacggta aatggcccac ttggcagtac atcaatatct attaatagta
      721 acttggcaag tacattacta ttggaagtac gccagggtac attggcagta ctcccattga
      781 cgtcaatggc ggtaaatggc ccgcgatggc tgccaagtac atccccattg acgtcaatgg
      841 ggaggggcaa tgacgcaaat gggcgttcca ttgacgtaaa tgggcggtag gcgtgcctaa
      901 tgggaggtct atataagcaa tgctcgttta gggaaccgcc attctgcctg gggacgtcgg
      961 agcaagcttg atttaggtga cactatagag gtctcaacta ccggatccgc cgccatggct
     1021 ccaaagaaga agcgtaaggt aatggtgagc aagggcgagg aggacaacat ggccatcatc
     1081 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggccg cgagttcgag
     1141 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     1201 accaagggcg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
     1261 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
     1321 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     1381 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
     1441 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag
     1501 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
     1561 gacggcggcc actacgacgc cgaggtcaag accacctaca aggccaagaa gcccgtgcag
     1621 ctgcccggcg cctacaacgt caacatcaag ctggacatca cctcccacaa cgaggactac
     1681 accatcgtgg aacagtacga gcgcgccgag ggccgccact ccaccggcgg catggacgag
     1741 ctgtacaagg attacaagga tgacgatgac aagtagggta cctaagtgag accctcatcc
     1801 tcccgggtaa ctaagtaagg atctcgaccg atcctgagaa cttcagggtg agtttgggga
     1861 cccttgattg ttctttcttt ttcgctattg taaaattcat gttatatgga gggggcaaag
     1921 ttttcagggt gttgtttaga atgggaagat gtcccttgta tcaccatgga ccctcatgat
     1981 aattttgttt ctttcacttt ctactctgtt gacaaccatt gtctcctctt attttctttt
     2041 cattttctgt aactttttcg ttaaacttta gcttgcattt gtaacgaatt tttaaattca
     2101 cttttgttta tttgtcagat tgtaagtact ttctctaatc actttttttt caaggcaatc
     2161 agggtatatt atattgtact tcagcacagt tttagagaac aattgttata attaaatgat
     2221 aaggtagaat atttctgcat ataaattctg gctggcgtgg aaatattctt attggtagaa
     2281 acaactacac cctggtcatc atcctgcctt tctctttatg gttacaatga tatacactgt
     2341 ttgagatgag gataaaatac tctgagtcca aaccgggccc ctctgctaac catgttcatg
     2401 ccttcttctc tttcctacag ctcctgggca acgtgctggt tgttgtgctg tctcatcatt
     2461 ttggcaaaga attaattcct cgacggatcg tagatccaga catgataaga tacattgatg
     2521 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     2581 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     2641 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa ttcgcggccg
     2701 ctctagaact agtctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2761 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     2821 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     2881 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2941 gctggcgttt ttccataggc tcggcccccc tgacgagcat cacaaaaatc gacgctcaag
     3001 tcagaggtgg cgaaacccga caggactata aagataccag gcgttccccc ctggaagctc
     3061 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3121 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt
     3181 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3241 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3301 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3361 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     3421 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3481 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3541 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3601 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3661 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     3721 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     3781 gcccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     3841 gataccgcgg gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     3901 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     3961 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4021 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4081 ccaacgatca aggcgagtta catgatcccc catgttgtga aaaaaagcgg ttagctcctt
     4141 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tgcttatggc
     4201 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     4261 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     4321 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     4381 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     4441 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     4501 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     4561 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     4621 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     4681 tccccgaaaa gtgccacctg
//
