LOCUS       sequence-205158-        3856 bp    DNA     linear   UNA 14-APR-2025
DEFINITION  T7 promotor drives in vitro mRNA transcription of a mEGFP with a 
            nuclear localisation signal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 3856)
  AUTHORS   Clift D, McEwan WA, Labzin LI, Konieczny V, Mogessie B, James LC, 
            Schuh M
  TITLE     A Method for the Acute and Rapid Degradation of Endogenous Proteins.
  JOURNAL   Cell. 2017 Nov 13. pii: S0092-8674(17)31255-2. doi: 
            10.1016/j.cell.2017.10.033.
  PUBMED    29153837
REFERENCE   2  (bases 1 to 3856)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2017 
            Nov 13. pii: S0092-8674(17)31255-2. doi: 
            10.1016/j.cell.2017.10.033."
COMMENT     Sequence Label: pGEMHE-NLS-mEGFP
FEATURES             Location/Qualifiers
     source          1..3856
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        complement(59..77)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(60..77)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(95..111)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(95..111)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(108..130)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    119..135
                     /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(143..149,150..167,168..173))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    188..209
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(326..343)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(497..1085)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(577..596)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1256..2047,2048..2116))
                     /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     1879..1898
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2117..2221)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2289..2307
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2345..2367)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2467..2486
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2680..2702
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2694..2711
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2695..2711
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2718..2737
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2718..2736
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           2768..2810
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             2837..2884
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     regulatory      2897..2906
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2903..2905,2906..2908,2909..3619)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2948..2969)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3209..3228)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     3'UTR           3728..3853
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
ORIGIN
        1 cccccccccc tgcaggcgcg ccgccggcga tttaaattaa ttaatctagc ttgagtattc
       61 tatagtgtca cctaaatagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
      121 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
      181 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
      241 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
      301 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
      361 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
      421 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
      481 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
      541 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
      601 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
      661 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
      721 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
      781 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
      841 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
      901 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
      961 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1021 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1081 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1141 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     1201 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     1261 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     1321 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     1381 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     1441 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     1501 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     1561 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     1621 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     1681 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     1741 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     1801 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     1861 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     1921 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     1981 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2041 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     2101 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     2161 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     2221 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     2281 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     2341 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     2401 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     2461 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     2521 acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact
     2581 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     2641 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     2701 cgacggccag tgaattgtaa tacgactcac tatagggcga attaattcga gctcggtacc
     2761 cagcttgctt gttctttttg cagaagctca gaataaacgc tcaactttgg cagatcaatt
     2821 ccccggtgct agcatgaagc ggcccgctgc tactaagaag gctggtcagg ctaagaagaa
     2881 gaagcttcca ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
     2941 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     3001 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     3061 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     3121 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     3181 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     3241 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     3301 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     3361 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     3421 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     3481 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     3541 cgagaagcgc gatcacatgg tccttaagga gttcgtgacc gccgccggga tcactctcgg
     3601 catggacgag ctgtacaagt actcagatct cgagctcaag cttcgaattc tgcagtcgac
     3661 ggcccgggat cctgtacaag taaagcggcc gctctagata aatgatagct tgatctggtt
     3721 accactaaac cagcctcaag aacacccgaa tggagtctct aagctacata ataccaactt
     3781 acactttaca aaatgttgtc ccccaaaatg tagccattcg tatctgctcc taataaaaag
     3841 aaagtttctt cacatt
//
