LOCUS       sequence-99754-e        7905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Red click beetle luciferase (Promega) driven by LEU1 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7905)
  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 7905)
  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: pNB777
FEATURES             Location/Qualifiers
     source          1..7905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(340..359)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(341..359)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(369..386)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(369..385)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(378..400)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      530..985
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(617..636)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     827..848
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1116..1919)
                     /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(1920..2140)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(2166..2185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2285..2307
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2345..2363)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2431..2535
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2536..2604,2605..3396)
                     /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(2754..2773)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3567..4155
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4056..4075
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4309..4326
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(4479..4484,4485..4502,4503..4509)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4517..4533
                     /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     4522..4544
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4541..4557
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4541..4557
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4576..4596
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4578..4596
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             5519..5530
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             6030..7655
                     /codon_start=1
                     /product="red-emitting variant of click beetle luciferase"
                     /label=CBRluc
                     /note="human codon-optimized"
                     /translation="MVKREKNVIYGPEPLHPLEDLTAGEMLFRALRKHSHLPQALVDVV
                     GDESLSYKEFFEATVLLAQSLHNCGYKMNDVVSICAENNTRFFIPVIAAWYIGMIVAPV
                     NESYIPDELCKVMGISKPQIVFTTKNILNKVLEVQSRTNFIKRIIILDTVENIHGCESL
                     PNFISRYSDGNIANFKPLHFDPVEQVAAILCSSGTTGLPKGVMQTHQNICVRLIHALDP
                     RYGTQLIPGVTVLVYLPFFHAFGFHITLGYFMVGLRVIMFRRFDQEAFLKAIQDYEVRS
                     VINVPSVILFLSKSPLVDKYDLSSLRELCCGAAPLAKEVAEVAAKRLNLPGIRCGFGLT
                     ESTSAIIQTLGDEFKSGSLGRVTPLMAAKIADRETGKALGPNQVGELCIKGPMVSKGYV
                     NNVEATKEAIDDDGWLHSGDFGYYDEDEHFYVVDRYKELIKYKGSQVAPAELEEILLKN
                     PCIRDVAVVGIPDLEAGELPSAFVVKQPGTEITAKEVYDYLAERVSHTKYLRGGVRFVD
                     SIPRNVTGKITRKELLKQLLVKAGG"
     RBS             6980..6988
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     terminator      7688..7875
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(7743..7767)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
ORIGIN
        1 gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg gaggcccaga
       61 ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt
      121 acatttagcc catacatccc catgtataat catttgcatc catacatttt gatggccgca
      181 cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga aacgctcccc
      241 tcacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg
      301 acgctcgaag gctttaattt gcggccggta cccaattcgc cctatagtga gtcgtattac
      361 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
      421 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
      481 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcga cgcgccctgt
      541 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
      601 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
      661 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
      721 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
      781 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
      841 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
      901 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
      961 aacaaaatat taacgtttac aatttcctga tgcggtattt tctccttacg catctgtgcg
     1021 gtatttcaca ccgcataggg taataactga tataattaaa ttgaagctct aatttgtgag
     1081 tttagtatac atgcatttac ttataataca gttttttagt tttgctggcc gcatcttctc
     1141 aaatatgctt cccagcctgc ttttctgtaa cgttcaccct ctaccttagc atcccttccc
     1201 tttgcaaata gtcctcttcc aacaataata atgtcagatc ctgtagagac cacatcatcc
     1261 acggttctat actgttgacc caatgcgtct cccttgtcat ctaaacccac accgggtgtc
     1321 ataatcaacc aatcgtaacc ttcatctctt ccacccatgt ctctttgagc aataaagccg
     1381 ataacaaaat ctttgtcgct cttcgcaatg tcaacagtac ccttagtata ttctccagta
     1441 gatagggagc ccttgcatga caattctgct aacatcaaaa ggcctctagg ttcctttgtt
     1501 acttcttctg ccgcctgctt caaaccgcta acaatacctg ggcccaccac accgtgtgca
     1561 ttcgtaatgt ctgcccattc tgctattctg tatacacccg cagagtactg caatttgact
     1621 gtattaccaa tgtcagcaaa ttttctgtct tcgaagagta aaaaattgta cttggcggat
     1681 aatgccttta gcggcttaac tgtgccctcc atggaaaaat cagtcaagat atccacatgt
     1741 gtttttagta aacaaatttt gggacctaat gcttcaacta actccagtaa ttccttggtg
     1801 gtacgaacat ccaatgaagc acacaagttt gtttgctttt cgtgcatgat attaaatagc
     1861 ttggcagcaa caggactagg atgagtagca gcacgttcct tatatgtagc tttcgacatg
     1921 atttatcttc gtttcctgca ggtttttgtt ctgtgcagtt gggttaagaa tactgggcaa
     1981 tttcatgttt cttcaacact acatatgcgt atatatacca atctaagtct gtgctccttc
     2041 cttcgttctt ccttctgttc ggagattacc gaatcaaaaa aatttcaaag aaaccgaaat
     2101 caaaaaaaag aataaaaaaa aaatgatgaa ttgaattgaa aagctgtggt atggtgcact
     2161 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     2221 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     2281 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     2341 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     2401 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2461 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2521 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2581 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2641 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2701 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2761 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     2821 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2881 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2941 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3001 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3061 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3121 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3181 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3241 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3301 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3361 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3421 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3481 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3541 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3601 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3661 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3721 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3781 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3841 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3901 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3961 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4021 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4081 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4141 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4201 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4261 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4321 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     4381 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     4441 attaatgtga gttacctcac tcattaggca ccccaggctt tacactttat gcttccggct
     4501 cctatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     4561 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga gctctgatga
     4621 acggtaagcc aatgaaggaa aagaagtcta ccagaagtgt cgaagacttc atggctgcta
     4681 tgcaaagagt ctctactcaa cacgaaaagg aaacctaatc ctgttgaagt agcatttaat
     4741 cataattttt gtcacatttt aatcaacttg atttttctgg tttaattttt ctaattttaa
     4801 ttttaatttt tttatcaatg ggaactgata cactaaaaag aattaggagc caacaagaat
     4861 aagccgctta tttcctacta gagtttgctt aaaatttcat ctcgaattgt cattctaata
     4921 ttttatccac acacacacct taaaattttt agattaaatg gcatcaactc ttagcttcac
     4981 acacacacac acaccgaagc tggttgtttt atttgatttg atataattgg tttctctgga
     5041 tggtactttt tctttcttgg ttatttccta ttttaaaata tgaaacgcac acaagtcata
     5101 attattctaa tagagcacaa ttcacaacac gcacatttca actttaatat ttttttagaa
     5161 acactttatt tagtctaatt cttaattttt aatatatata atgcacacac actaatttat
     5221 tcattaattt tttattgagt aggatttgaa aatatttggt atctttgcaa gatgtttgta
     5281 tagagggaca aagaatcgtc tttattatgg tcaaggcttt acgtcataat agttcctgcc
     5341 cagctcttct ataatacttt aaagatctct tctcgtttgc tccatttgga agtctcgctt
     5401 acgtttatgc gcccatacag acactcaaga tacacactta catgaacgta tacaaattta
     5461 ctaacactac ttgaaaatat gaaccacagt acatcatatt aagacgtagt attcgatgat
     5521 tgaaggccgc ctccgcgaaa tacctttact gattttgccg gttaatcgca tcgaaatttc
     5581 ttcatcacaa gaaagcaaac aaatcgccag gccattctac aagtttcctt ttcttatgaa
     5641 gatgtaaaag ctactaaggc gtcattactc tagatgactc agtttagtct gaccttctat
     5701 agtatactac cctggcgcta tgatgatgag cggttctttt attgcggaaa cgaaaattcc
     5761 gggaccggcg aaatttgccc ggttttgtcc gtaaccggct tcatgagtcg gcttcaatag
     5821 tagttgaata cttatttaaa cagcagaact taactcactc atcacgctgt ttccgctgaa
     5881 ttttctcaaa atatctaagc agtcaacaaa tataaagaat attgaaattg acagtttttg
     5941 tcgctatcga tttttattat ttgctgtttt aaatcttggt ttacactcca tccaaactag
     6001 aactagtgga tcccccgggt taattaacca tggtaaagcg tgagaaaaat gtcatctatg
     6061 gccctgagcc tctccatcct ttggaggatt tgactgccgg cgaaatgctg tttcgtgctc
     6121 tccgcaagca ctctcatttg cctcaagcct tggtcgatgt ggtcggcgat gaatctttga
     6181 gctacaagga gttttttgag gcaaccgtct tgctggctca gtccctccac aattgtggct
     6241 acaagatgaa cgacgtcgtt agtatctgtg ctgaaaacaa tacccgtttc ttcattccag
     6301 tcatcgccgc atggtatatc ggtatgatcg tggctccagt caacgagagc tacattcccg
     6361 acgaactgtg taaagtcatg ggtatctcta agccacagat tgtcttcacc actaagaata
     6421 ttctgaacaa agtcctggaa gtccaaagcc gcaccaactt tattaagcgt atcatcatct
     6481 tggacactgt ggagaatatt cacggttgcg aatctttgcc taatttcatc tctcgctatt
     6541 cagacggcaa catcgcaaac tttaaaccac tccacttcga ccctgtggaa caagttgcag
     6601 ccattctgtg tagcagcggt actactggac tcccaaaggg agtcatgcag acccatcaaa
     6661 acatttgcgt gcgtctgatc catgctctcg atccacgcta cggcactcag ctgattcctg
     6721 gtgtcaccgt cttggtctac ttgcctttct tccatgcttt cggctttcat attactttgg
     6781 gttactttat ggtcggtctc cgcgtgatta tgttccgccg ttttgatcag gaggctttct
     6841 tgaaagccat ccaagattat gaagtccgca gtgtcatcaa cgtgcctagc gtgatcctgt
     6901 ttttgtctaa gagcccactc gtggacaagt acgacttgtc ttcactgcgt gaattgtgtt
     6961 gcggtgccgc tccactggct aaggaggtcg ctgaagtggc cgccaaacgc ttgaatcttc
     7021 cagggattcg ttgtggcttc ggcctcaccg aatctaccag tgcgattatc cagactctcg
     7081 gggatgagtt taagagcggc tctttgggcc gtgtcactcc actcatggct gctaagatcg
     7141 ctgatcgcga aactggtaag gctttgggcc cgaaccaagt gggcgagctg tgtatcaaag
     7201 gccctatggt gagcaagggt tatgtcaata acgttgaagc taccaaggag gccatcgacg
     7261 acgacggctg gttgcattct ggtgattttg gatattacga cgaagatgag catttttacg
     7321 tcgtggatcg ttacaaggag ctgatcaaat acaagggtag ccaggttgct ccagctgagt
     7381 tggaggagat tctgttgaaa aatccatgca ttcgcgatgt cgctgtggtc ggcattcctg
     7441 atctggaggc cggcgaactg ccttctgctt tcgttgtcaa gcagcctggt acagaaatta
     7501 ccgccaaaga agtgtatgat tacctggctg aacgtgtgag ccatactaag tacttgcgtg
     7561 gcggcgtgcg ttttgttgac tccatccctc gtaacgtaac aggcaaaatt acccgcaagg
     7621 agctgttgaa acaattgttg gtgaaggccg gcggtttgta caaataaggc gcgccacttc
     7681 taaataagcg aatttcttat gatttatgat ttttattatt aaataagtta taaaaaaaat
     7741 aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct tattcttgag
     7801 taactctttc ctgtaggtca ggttgctttc tcaggtatag tatgaggtcg ctcttattga
     7861 ccacacctct accggcagat ccgctaggga taacagggta atata
//
