LOCUS       sequence-481962-        6023 bp    DNA     circular SYN 04-AUG-2025
DEFINITION  Express CCNE1-NanoLuc Fusion Protein in Mammalian Cells under a CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6023)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6023)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: CCNE1-NanoLuc Vector
FEATURES             Location/Qualifiers
     source          1..6023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    17..98
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     42..61
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(275..787)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     promoter        complement(2077..2095)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2078..2095)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        2215..2518
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2673..2693
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        2726..2773
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2805..3599
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2859..2878)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3469..3488
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(3757..4345)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3837..3856)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4471..4519
                     /note="synthetic polyadenylation signal"
     CDS             complement(4543..5580)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4556..4564
                     /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)"
     promoter        complement(5611..5968)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5625..5760
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5777..5968
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5925..5945
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 aagacgcgtg acgtcggcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
       61 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
      121 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
      181 aatgtggtat ggctgattat gatccgcggc cgcaatacgt cgacgttatc agctgacttc
      241 gtacgagagc ctaggtgttt ctagagtcgc ggccttacgc cagaatgcgt tcgcacagcc
      301 gccagccggt cactccgttg atggttactc ggaacagcag ggagccgtcg gggttgatca
      361 ggcgctcgtc gataattttg ttgccgttcc acagggtccc tgttacagtg atctttttgc
      421 cgtcgaacac ggcgatgcct tcatacggcc gtccgaaata gtcgatcatg ttcggcgtaa
      481 ccccgtcgat taccagtgtg ccatagtgca ggatcacctt aaagtgatga tcatccacag
      541 ggtacaccac cttaaaaatt ttttcgatct ggcccatttg gtcgccgctc agaccttcat
      601 acgggatgat gacatggatg tcgatcttca gcccattttc accgctcagg acaatccttt
      661 ggatcggagt tacggacacc ccgagattct gaaacaaact ggacacacct ccctgttcaa
      721 ggacttggtc caggttgtag ccggctgtct gtcgccagtc cccaacgaaa tcttcgagtg
      781 tgaagacgcc gctcgagccg agagaaaccg ccatttccgg cccgctgctc tgcttcttac
      841 cgctctgtgg cggggtgagg agcccactgg ggagaggaga agccctattt tgttcagaca
      901 acatggcttt ctttgctcgg gctttgtcca gcaaatccaa gctgtctctg tgggtctgta
      961 tgttgtgtgc atcttcatca gcgacgcccc tgaagtgctt cagttttgag ctccccgtct
     1021 cccttataac catggcaaat ggaaccatcc acttgacaca gttctctatg tcgcaccact
     1081 gataccctga aaccttttgc atcaattcag atgacgagaa atgatacaag gccgaagcag
     1141 caagtatacc ataaggaaat tcaaggcagt caacatccag gacacagaga tccaacagct
     1201 ctgcaatctg tataaagatt tgctggggat actgcggcag tagcacttca tgtaagtcat
     1261 ttagatatgc aacctgcatg tatacattca gccaggacac aatagtcagg ggacttaaac
     1321 gccacttaag ggccttcata atcattaatt ccatggtgag aatttcatct cctgaacaag
     1381 ctccatctgt cacatacgca aactggtgca actttggagg atagatttcc tcaagtttgg
     1441 ctgcaataaa taaagatgaa atcccaataa gctgtaaaag agtttttaca acattttctt
     1501 gtgtcgccat ataccggtca aagaaatctt gtgccaagta aaaggtctcc ctgtgaagtt
     1561 tatagacttc acacacctcc attaaccaat ccagaagaat tgctcgcatt tttggctgca
     1621 gaagagggtg ttgctcaaga aagtgctgat cccttaagta tgtcttttcc ttgtttaaca
     1681 tgattttcca gacttcctct ctatttgccc agctcagtac aggcagcggg gagcctctgg
     1741 atggtgcaat aatccgaggc ttgcacgttg agtttgggta aacccggtca tcatcttctt
     1801 tgtcaggtgt ggggatcagg gagcaggggt ctgcacagac tgcattattg tcccaaggct
     1861 ggctcccaca ctggtccctc gccgtcctgt cgattttggc catttcttca tctggatcct
     1921 gcaaaaaaac ggtcacgttt gccttcctct tcctggagcg agccgagaac tccgcgccgc
     1981 cgtcctcctt catggtgtcc cgctccttcg catcccgctc cctgcgctcc ctcggcatgg
     2041 cgatcgctag ctgcaaaaag aacaagtagc ttgtattcta tagtgtcacc taaatgggcg
     2101 atctgacggt tcactaaacc agctctgctt atataggcct ccctggcgcg ccactagtta
     2161 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     2221 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     2281 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     2341 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtcc
     2401 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     2461 cttacgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     2521 gatgcggttt tggcagtaca ccaatgggcg tggatagcgg tttgactcac ggggatttcc
     2581 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     2641 tccaaaatgt cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg
     2701 ggaggtcaaa tcgaattcta atactgttga caattaatca tcggcatagt atatcggcat
     2761 agtataatac gacaaggtga ggaactaaac ccaggaggca gatcatgatt gaacaagatg
     2821 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     2881 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     2941 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     3001 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     3061 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     3121 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     3181 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     3241 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     3301 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     3361 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     3421 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     3481 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     3541 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgat
     3601 tcgaaatgac cgaccaagcg acgcccaacc ggtatcagct cactcaaagg cggtaatacg
     3661 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3721 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3781 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3841 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3901 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3961 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4021 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4081 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4141 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     4201 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4261 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4321 tacgcgcaga aaaaaaggat ttcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4381 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4441 cacctagatc cttttatagt ccggaatcgt cacacaaaaa accaacacac agatgtaatg
     4501 aaaataaaga tattttattg cggccatcgt gatggctaga gtctaaactc gacctacctc
     4561 cttagcgcgc ggtcgtgtac tgggccggcg gttgccgctg tcggccagca cctcgacgca
     4621 gccgtcggtc catacggctg cgctccggcg agcgatttgt gtgcgaccga cggtgccggc
     4681 cccgctgcgg acgatggcat cgcagcggcc ttgagcccag gcagcatcgt cgaagttgcc
     4741 gtccacgagg ctctggtaga gctgatccag gccgatgcgc agcatgtagg ctcgcagacg
     4801 agggctgccg gccagctcgg gatgccggcg ctcgaagtag cgagtctgct gctccatgca
     4861 agccagccag ggccgccaga agaagatgtt ggccacctcg tactgactgt ccccgaacat
     4921 agcttcggac cagtcgatta cggcggtgat gcggccgttg tcggtcagga cgttgttgct
     4981 gccgaagtcg gcatggacga ggtggcgcac ctcgggacag tcttcggccc acagcatcag
     5041 ttcgtccagg gcttgagcta cgctggcgga cacggtgtcg tccatcacgg tctgccagtg
     5101 gtagacatgg ggatcagcaa tggcgcaaat gaaatcccgc caggttgtgt actggccgat
     5161 gccttggggc ccgaatgggc cgaagccgct ggtttgactg aggtcggcgg cggcaatagc
     5221 atccatagct tcggcgacag gctgtaacac agcaggcagc tctgtttcgg ggaggtcttg
     5281 gagagtaacg ccttgggcgc gtctactgat gcagtatgtc aggctctcgc tgaactcgcc
     5341 gatgtccaac acttcgggga tgggtagtgc agcgctggcg aagtggcggt acacgtagcg
     5401 gtctttgtag aagccatcag cgcagctatt cacccgcagt acatagccgc gtccgccgac
     5461 atcgaagctg aaggctcggc tctcttcgcc ctccgacaac tgcatcaggt cgctcacact
     5521 gtcgaacttc tcgatgagaa atttttcaac gctggtagcg gtgagttcgg gcttcttcat
     5581 ggtggcgcta gtgtcagaag aatcgagctt tttgcaaaag cctaggcctc caaaaaagcc
     5641 tcctcactac ttctggaata gctcagaggc cgaggcggcc tcggcctctg cataaataaa
     5701 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat
     5761 gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact
     5821 tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat
     5881 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac
     5941 attccacagc tggttctttc cgcctcagaa ggtacctaac caagttcctc tttcagaggt
     6001 tatttcaggc catggtgctg cgc
//
