LOCUS       sequence-248775-        7074 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The expressed ORF encodes nucleus location signal with GFP and 
            destabilized mutant Luciferase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7074)
  AUTHORS   Guo L, Prall W, Yang X
  TITLE     Assays for the Degradation of Misfolded Proteins in Cells.
  JOURNAL   J Vis Exp. 2016 Aug 28;(114). doi: 10.3791/54266.
  PUBMED    27684355
REFERENCE   2  (bases 1 to 7074)
  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: "J Vis 
            Exp."; date: "2016-08-28"; volume: "(114). doi"; pages: " 
            10.3791/54266"
COMMENT     Sequence Label: NLS-LucDM-GFP
FEATURES             Location/Qualifiers
     source          1..7074
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        1020..1399
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1400..1603
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1554..1574
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1600..1624
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1834..1852
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1834..1851
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1865..1884
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1931..1940
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1940..1963
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1970..1990
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2054..2072)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             join(3656..3658,3659..4372)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3656..3658,3659..4369)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3698..3719)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3959..3978)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4306..4327
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      4380..4389
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4393..4527
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4430..4449)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4484..4503
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4596..4953
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4596..4787
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4619..4639)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4804..4939
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4973..4990)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4973..4989)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4982..5004)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5202..5657
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5289..5308)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5499..5520
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5674..5693)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5793..5815
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5853..5871)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5939..6043
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6044..6112,6113..6904)
                     /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     complement(6262..6281)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga catgattacg aattaattcg agctcgcccg
     1021 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     1081 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     1141 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     1201 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     1261 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     1321 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     1381 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     1441 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     1501 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     1561 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca
     1621 gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc
     1681 cagcctccgc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca agagtgacgt
     1741 aagtaccgcc tatagagtct ataggcccac ccccttggct tcgttagaac gcggctacaa
     1801 ttaatacata accttatgta tcatacacat acgatttagg tgacactata gaataacatc
     1861 cactttgcct ttctctccac aggtgtccac tcccaggtcc aactgcacct cggttctatc
     1921 gattgaattc gccgccatgg actacaagga cgacgatgac aagggatccc ctaagaagaa
     1981 gcgaaaggtt gaagacgcca aaaacataaa gaaaggcccg gcgccattct atccgctgga
     2041 agatggaacc gctggagagc aactgcataa ggctatgaag agatacgccc tggttcctgg
     2101 aacaattgct tttacagatg cacatatcga ggtggacatc acttacgctg agtacttcga
     2161 aatgtccgtt cggttggcag aagctatgaa acgatatggg ctgaatacaa atcacagaat
     2221 cgtcgtatgc agtgaaaact ctcttcaatt ctttatgccg gtgttgggcg cgttatttat
     2281 cggagttgca gttgcgcccg cgaacgacat ttataatgaa cgtgaattgc tcaacagtat
     2341 gggcatttcg cagcctaccg tggtgttcgt ttccaaaaag gggttgcaaa aaattttgaa
     2401 cgtgcaaaaa aagctcccaa tcatccaaaa aattattatc atggattcta aaacggatta
     2461 ccagggattt cagtcgatgt acacgttcgt cacatctcat ctacctcccg gttttaatga
     2521 atacgatttt gtgccagagt ccttcgatca ggacaagaca attgcactga tcatgaactc
     2581 ctctggatct actggtctgc ctaaaggtgt cgctctgcct catagaactg cctgcgtgag
     2641 attctcgcat gccagagatc ctatttttgg caatcaaatc attccggata ctgcgatttt
     2701 aagtgttgtt ccattccatc acggttttgg aatgtttact acactcggat atttgatatg
     2761 tggatttcaa gtcgtcttaa tgtatagatt tgaagaagag ctgtttctga ggagccttca
     2821 ggattacaag attcaaagtg cgctgctggt gccaacccta ttctccttct tcgccaaaag
     2881 cactctgatt gacaaatacg atttatctaa tttacacgaa attgcttctg gtggcgctcc
     2941 cctctctaag gaagtcgggg aagcggttgc caagaggttc catctgccag gtatcaggca
     3001 aggatatggg ctcactgaga ctacatcagc tattctgatt acacccgagg gggatgataa
     3061 accgggcgcg gtcggtaaag ttgttccatt ttttgaagcg aaggttgtgg atctggatac
     3121 cgggaaaacg ctgggcgtta atcaaagagg cgaactgtgt gtgagaggtc ctatgattat
     3181 gtccggttat gtaaacaatc cggaagcgac caacgccttg attgacaagg atggatggct
     3241 acattctgga gacatagctt actgggacga agacgaacac ttcttcatcg ttgaccgcct
     3301 gaagtctctg attaagtaca aaggctatca ggtggctccc gctgaattgg aatccatctt
     3361 gctccaacac cccaacatct tcgacgcagg tgtcgcaggt cttcccgacg atgacgccgg
     3421 tgaacttccc gccgccgttg ttgttttgga gcacggaaag acgatgacgg aaaaagagat
     3481 cgtggattac gtcgccagtc aagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtt
     3541 tgtggacgaa gtaccgaaag gtcttaccgg aaaactcgac gcaagaaaaa tcagagagat
     3601 cctcataaag gccaagaagg gcggaaagtc caaaatcgtc gacaccatgt ctagagtgag
     3661 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     3721 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     3781 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     3841 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     3901 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     3961 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     4021 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     4081 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     4141 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     4201 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     4261 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     4321 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aaaagcttgg
     4381 ccgccatggc ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
     4441 acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     4501 atcaatgtat cttatcatgt ctggatcggg aattaattcg gcgcagcacc atggcctgaa
     4561 ataacctctg aaagaggaac ttggttaggt accttctgag gcggaaagaa ccagctgtgg
     4621 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4681 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     4741 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4801 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4861 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     4921 ggaggctttt ttggaggcct aggcttttgc aaaaagctgt taacagcttg gcactggccg
     4981 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     5041 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     5101 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     5161 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
     5221 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     5281 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     5341 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     5401 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     5461 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     5521 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc
     5581 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     5641 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     5701 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5761 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5821 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
     5881 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5941 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     6001 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     6061 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     6121 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     6181 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     6241 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     6301 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     6361 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6421 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6481 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6541 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6601 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6661 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6721 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6781 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6841 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6901 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6961 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     7021 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttc
//
