LOCUS       sequence-96210-e        5840 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NanoLuc luciferase with a nuclear localization signal (NLS) driven 
            by MET17 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5840)
  AUTHORS   Mazo-Vargas A, Park H, Aydin M, Buchler NE
  TITLE     Measuring fast gene dynamics in single cells with time-lapse 
            luminescence microscopy
  JOURNAL   Mol Biol Cell. 2014 Sep 17. pii: mbc.E14-07-1187.
  PUBMED    25232010
REFERENCE   2  (bases 1 to 5840)
  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: "Mol Biol 
            Cell. 2014 Sep 17. pii: mbc.E14-07-1187."
COMMENT     Sequence Label: pNB648
FEATURES             Location/Qualifiers
     source          1..5840
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /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(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /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     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2241..2261
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2243..2261
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2312..2661
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     primer_bind     2662..2678
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2662..2678
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             2707..2727
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2731..3243
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     terminator      3288..3475
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3343..3367)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3845..3864)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3846..3864)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3874..3891)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3874..3890)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3883..3905)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4035..4490
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4122..4141)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4332..4353
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4621..5424)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(5425..5645)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5671..5690)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5790..5812
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttac ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcctat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctcc
     2281 ggatgcaagg gttcgaatcc cttagctctc attatttttt gctttttctc ttgaggtcac
     2341 atgatcgcaa aatggcaaat ggcacgtgaa gctgtcgata ttggggaact gtggtggttg
     2401 gcaaatgact aattaagtta gtcaaggcgc catcctcatg aaaactgtgt aacataataa
     2461 ccgaagtgtc gaaaaggtgg caccttgtcc aattgaacac gctcgatgaa aaaaataaga
     2521 tatatataag gttaagtaaa gcgtctgtta gaaaggaagt ttttcctttt tcttgctctc
     2581 ttgtcttttc atctactatt tccttcgtgt aatacagggt cgtcagatac atagatacaa
     2641 ttctattacc cccatccata ctctagaact agtggatccc ccgggttaat taacaccatg
     2701 gcaccaccaa agaaaaagag aaaagtagcc atggtcttca cactcgaaga tttcgttggg
     2761 gactggcgac agacagccgg ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc
     2821 agtttgtttc agaatctcgg ggtgtccgta actccgatcc aaaggattgt cctgagcggt
     2881 gaaaatgggc tgaagatcga catccatgtc atcatcccgt atgaaggtct gagcggcgac
     2941 caaatgggcc agatcgaaaa aatttttaag gtggtgtacc ctgtggatga tcatcacttt
     3001 aaggtgatcc tgcactatgg cacactggta atcgacgggg ttacgccgaa catgatcgac
     3061 tatttcggac ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca
     3121 gggaccctgt ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc cgacggctcc
     3181 ctgctgttcc gagtaaccat caacggagtg accggctggc ggctgtgcga acgcattctg
     3241 gcgggtgggg gatcattgta caaataaggc gcgccacttc taaataagcg aatttcttat
     3301 gatttatgat ttttattatt aaataagtta taaaaaaaat aagtgtatac aaattttaaa
     3361 gtgactctta ggttttaaaa cgaaaattct tattcttgag taactctttc ctgtaggtca
     3421 ggttgctttc tcaggtatag tatgaggtcg ctcttattga ccacacctct accggcagat
     3481 ccgctaggga taacagggta atatagatct gtttagcttg cctcgtcccc gccgggtcac
     3541 ccggccagcg acatggaggc ccagaatacc ctccttgaca gtcttgacgt gcgcagctca
     3601 ggggcatgat gtgactgtcg cccgtacatt tagcccatac atccccatgt ataatcattt
     3661 gcatccatac attttgatgg ccgcacggcg cgaagcaaaa attacggctc ctcgctgcag
     3721 acctgcgagc agggaaacgc tcccctcaca gacgcgtgta cgcatgtaac attatactga
     3781 aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcggc cggtacccaa
     3841 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     3901 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     3961 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4021 tggcgaatgg cgcgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4081 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4141 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4201 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     4261 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4321 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     4381 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     4441 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt cctgatgcgg
     4501 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tagggtaata actgatataa
     4561 ttaaattgaa gctctaattt gtgagtttag tatacatgca tttacttata atacagtttt
     4621 ttagttttgc tggccgcatc ttctcaaata tgcttcccag cctgcttttc tgtaacgttc
     4681 accctctacc ttagcatccc ttccctttgc aaatagtcct cttccaacaa taataatgtc
     4741 agatcctgta gagaccacat catccacggt tctatactgt tgacccaatg cgtctccctt
     4801 gtcatctaaa cccacaccgg gtgtcataat caaccaatcg taaccttcat ctcttccacc
     4861 catgtctctt tgagcaataa agccgataac aaaatctttg tcgctcttcg caatgtcaac
     4921 agtaccctta gtatattctc cagtagatag ggagcccttg catgacaatt ctgctaacat
     4981 caaaaggcct ctaggttcct ttgttacttc ttctgccgcc tgcttcaaac cgctaacaat
     5041 acctgggccc accacaccgt gtgcattcgt aatgtctgcc cattctgcta ttctgtatac
     5101 acccgcagag tactgcaatt tgactgtatt accaatgtca gcaaattttc tgtcttcgaa
     5161 gagtaaaaaa ttgtacttgg cggataatgc ctttagcggc ttaactgtgc cctccatgga
     5221 aaaatcagtc aagatatcca catgtgtttt tagtaaacaa attttgggac ctaatgcttc
     5281 aactaactcc agtaattcct tggtggtacg aacatccaat gaagcacaca agtttgtttg
     5341 cttttcgtgc atgatattaa atagcttggc agcaacagga ctaggatgag tagcagcacg
     5401 ttccttatat gtagctttcg acatgattta tcttcgtttc ctgcaggttt ttgttctgtg
     5461 cagttgggtt aagaatactg ggcaatttca tgtttcttca acactacata tgcgtatata
     5521 taccaatcta agtctgtgct ccttccttcg ttcttccttc tgttcggaga ttaccgaatc
     5581 aaaaaaattt caaagaaacc gaaatcaaaa aaaagaataa aaaaaaaatg atgaattgaa
     5641 ttgaaaagct gtggtatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     5701 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5761 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5821 gtcatcaccg aaacgcgcga
//
