LOCUS       sequence-481961-        6049 bp    DNA     circular SYN 04-AUG-2025
DEFINITION  Express NanoLuc-CCNE1 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 6049)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6049)
  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            "Unpublished"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: NanoLuc-CCNE1 Vector
FEATURES             Location/Qualifiers
     source          1..6049
                     /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(1552..2064)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     promoter        complement(2103..2121)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2104..2121)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        2241..2544
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2699..2719
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        2752..2799
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2831..3625
                     /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(2885..2904)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3495..3514
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(3783..4371)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3863..3882)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4497..4545
                     /note="synthetic polyadenylation signal"
     CDS             complement(4569..5606)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4582..4590
                     /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(5637..5994)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5651..5786
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5803..5994
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5951..5971
                     /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 ctagaggatc cccgggtacc gagcccgaat tcgtttaaac
      301 cgccatttcc ggcccgctgc tctgcttctt accgctctgt ggcggggtga ggagcccact
      361 ggggagagga gaagccctat tttgttcaga caacatggct ttctttgctc gggctttgtc
      421 cagcaaatcc aagctgtctc tgtgggtctg tatgttgtgt gcatcttcat cagcgacgcc
      481 cctgaagtgc ttcagttttg agctccccgt ctcccttata accatggcaa atggaaccat
      541 ccacttgaca cagttctcta tgtcgcacca ctgataccct gaaacctttt gcatcaattc
      601 agatgacgag aaatgataca aggccgaagc agcaagtata ccataaggaa attcaaggca
      661 gtcaacatcc aggacacaga gatccaacag ctctgcaatc tgtataaaga tttgctgggg
      721 atactgcggc agtagcactt catgtaagtc atttagatat gcaacctgca tgtatacatt
      781 cagccaggac acaatagtca ggggacttaa acgccactta agggccttca taatcattaa
      841 ttccatggtg agaatttcat ctcctgaaca agctccatct gtcacatacg caaactggtg
      901 caactttgga ggatagattt cctcaagttt ggctgcaata aataaagatg aaatcccaat
      961 aagctgtaaa agagttttta caacattttc ttgtgtcgcc atataccggt caaagaaatc
     1021 ttgtgccaag taaaaggtct ccctgtgaag tttatagact tcacacacct ccattaacca
     1081 atccagaaga attgctcgca tttttggctg cagaagaggg tgttgctcaa gaaagtgctg
     1141 atcccttaag tatgtctttt ccttgtttaa catgattttc cagacttcct ctctatttgc
     1201 ccagctcagt acaggcagcg gggagcctct ggatggtgca ataatccgag gcttgcacgt
     1261 tgagtttggg taaacccggt catcatcttc tttgtcaggt gtggggatca gggagcaggg
     1321 gtctgcacag actgcattat tgtcccaagg ctggctccca cactggtccc tcgccgtcct
     1381 gtcgattttg gccatttctt catctggatc ctgcaaaaaa acggtcacgt ttgccttcct
     1441 cttcctggag cgagccgaga actccgcgcc gccgtcctcc ttcatggtgt cccgctcctt
     1501 cgcatcccgc tccctgcgct ccctcggcat ggcgatcgcg ccgctcgagc ccgccagaat
     1561 gcgttcgcac agccgccagc cggtcactcc gttgatggtt actcggaaca gcagggagcc
     1621 gtcggggttg atcaggcgct cgtcgataat tttgttgccg ttccacaggg tccctgttac
     1681 agtgatcttt ttgccgtcga acacggcgat gccttcatac ggccgtccga aatagtcgat
     1741 catgttcggc gtaaccccgt cgattaccag tgtgccatag tgcaggatca ccttaaagtg
     1801 atgatcatcc acagggtaca ccaccttaaa aattttttcg atctggccca tttggtcgcc
     1861 gctcagacct tcatacggga tgatgacatg gatgtcgatc ttcagcccat tttcaccgct
     1921 caggacaatc ctttggatcg gagttacgga caccccgaga ttctgaaaca aactggacac
     1981 acctccctgt tcaaggactt ggtccaggtt gtagccggct gtctgtcgcc agtccccaac
     2041 gaaatcttcg agtgtgaaga ccatggtgag cgctagctgc aaaaagaaca agtagcttgt
     2101 attctatagt gtcacctaaa tgggcgatct gacggttcac taaaccagct ctgcttatat
     2161 aggcctccct ggcgcgccac tagttattaa tagtaatcaa ttacggggtc attagttcat
     2221 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
     2281 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     2341 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     2401 catcaagtgt atcatatgcc aagtccgccc cctattgacg tcaatgacgg taaatggccc
     2461 gcctggcatt atgcccagta catgacctta cgggactttc ctacttggca gtacatctac
     2521 gtattagtca tcgctattac catggtgatg cggttttggc agtacaccaa tgggcgtgga
     2581 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
     2641 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta ataaccccgc cccgttgacg
     2701 caaatgggcg gtaggcgtgt acggtgggag gtcaaatcga attctaatac tgttgacaat
     2761 taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaacccag
     2821 gaggcagatc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     2881 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     2941 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     3001 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     3061 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     3121 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     3181 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     3241 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     3301 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     3361 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     3421 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     3481 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     3541 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     3601 tcgccttctt gacgagttct tctgattcga aatgaccgac caagcgacgc ccaaccggta
     3661 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3721 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3781 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3841 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3901 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     3961 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4021 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4081 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4141 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4201 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     4261 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4321 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatttca agaagatcct
     4381 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4441 gtcatgagat tatcaaaaag gatcttcacc tagatccttt tatagtccgg aatcgtcaca
     4501 caaaaaacca acacacagat gtaatgaaaa taaagatatt ttattgcggc catcgtgatg
     4561 gctagagtct aaactcgacc tacctcctta gcgcgcggtc gtgtactggg ccggcggttg
     4621 ccgctgtcgg ccagcacctc gacgcagccg tcggtccata cggctgcgct ccggcgagcg
     4681 atttgtgtgc gaccgacggt gccggccccg ctgcggacga tggcatcgca gcggccttga
     4741 gcccaggcag catcgtcgaa gttgccgtcc acgaggctct ggtagagctg atccaggccg
     4801 atgcgcagca tgtaggctcg cagacgaggg ctgccggcca gctcgggatg ccggcgctcg
     4861 aagtagcgag tctgctgctc catgcaagcc agccagggcc gccagaagaa gatgttggcc
     4921 acctcgtact gactgtcccc gaacatagct tcggaccagt cgattacggc ggtgatgcgg
     4981 ccgttgtcgg tcaggacgtt gttgctgccg aagtcggcat ggacgaggtg gcgcacctcg
     5041 ggacagtctt cggcccacag catcagttcg tccagggctt gagctacgct ggcggacacg
     5101 gtgtcgtcca tcacggtctg ccagtggtag acatggggat cagcaatggc gcaaatgaaa
     5161 tcccgccagg ttgtgtactg gccgatgcct tggggcccga atgggccgaa gccgctggtt
     5221 tgactgaggt cggcggcggc aatagcatcc atagcttcgg cgacaggctg taacacagca
     5281 ggcagctctg tttcggggag gtcttggaga gtaacgcctt gggcgcgtct actgatgcag
     5341 tatgtcaggc tctcgctgaa ctcgccgatg tccaacactt cggggatggg tagtgcagcg
     5401 ctggcgaagt ggcggtacac gtagcggtct ttgtagaagc catcagcgca gctattcacc
     5461 cgcagtacat agccgcgtcc gccgacatcg aagctgaagg ctcggctctc ttcgccctcc
     5521 gacaactgca tcaggtcgct cacactgtcg aacttctcga tgagaaattt ttcaacgctg
     5581 gtagcggtga gttcgggctt cttcatggtg gcgctagtgt cagaagaatc gagctttttg
     5641 caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggccgag
     5701 gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg
     5761 gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac
     5821 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     5881 ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     5941 gactttccac accctaactg acacacattc cacagctggt tctttccgcc tcagaaggta
     6001 cctaaccaag ttcctctttc agaggttatt tcaggccatg gtgctgcgc
//
