LOCUS       sequence-72944-e        4757 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector: Position 2, HA tagged nls-EGFP with start 
            and stop codon.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4757)
  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 4757)
  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_2_+nls-eGFP-HA-_+1_BK+
FEATURES             Location/Qualifiers
     source          1..4757
                     /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             join(1087..1089,1090..1092,1093..1803)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1087..1089,1090..1092,1093..1803)
                     /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(1132..1153)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1393..1412)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1740..1761
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1804..1830
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     intron          1905..2477
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     polyA_signal    2550..2684
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2574..2593)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2628..2647
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2836..2853)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3007..3595)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(3766..4557,4558..4626))
                     /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     4389..4408
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4627..4731)
                     /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 gtctcaggct ccggatccgc cgccatggct
     1021 ccaaagaaga agcgtaaggt aaatcgatcg aattcaaggc ctctcgagcc tctagaaggt
     1081 ggcggaatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1141 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     1201 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1261 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     1321 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1381 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1441 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1501 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1561 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1621 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1681 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1741 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1801 aagtacccat acgatgttcc agattacgct tagggtacca ccttgagacc ctcatcctcc
     1861 cgggtaacta agtaaggatc tcgaccgatc ctgagaactt cagggtgagt ttggggaccc
     1921 ttgattgttc tttctttttc gctattgtaa aattcatgtt atatggaggg ggcaaagttt
     1981 tcagggtgtt gtttagaatg ggaagatgtc ccttgtatca ccatggaccc tcatgataat
     2041 tttgtttctt tcactttcta ctctgttgac aaccattgtc tcctcttatt ttcttttcat
     2101 tttctgtaac tttttcgtta aactttagct tgcatttgta acgaattttt aaattcactt
     2161 ttgtttattt gtcagattgt aagtactttc tctaatcact tttttttcaa ggcaatcagg
     2221 gtatattata ttgtacttca gcacagtttt agagaacaat tgttataatt aaatgataag
     2281 gtagaatatt tctgcatata aattctggct ggcgtggaaa tattcttatt ggtagaaaca
     2341 actacaccct ggtcatcatc ctgcctttct ctttatggtt acaatgatat acactgtttg
     2401 agatgaggat aaaatactct gagtccaaac cgggcccctc tgctaaccat gttcatgcct
     2461 tcttctcttt cctacagctc ctgggcaacg tgctggttgt tgtgctgtct catcattttg
     2521 gcaaagaatt aattcctcga cggatcgtag atccagacat gataagatac attgatgagt
     2581 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     2641 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     2701 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaattc gcggccgctc
     2761 tagaactagt ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2821 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2881 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2941 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3001 ggcgtttttc cataggctcg gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3061 gaggtggcga aacccgacag gactataaag ataccaggcg ttcccccctg gaagctccct
     3121 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3181 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3241 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3301 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3361 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3421 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     3481 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3541 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3601 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3661 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3721 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3781 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactgcc
     3841 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3901 accgcgggac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3961 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4021 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4081 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4141 acgatcaagg cgagttacat gatcccccat gttgtgaaaa aaagcggtta gctccttcgg
     4201 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgc ttatggcagc
     4261 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4321 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4381 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4441 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4501 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4561 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4621 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4681 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4741 ccgaaaagtg ccacctg
//
