LOCUS       sequence-478450-        6812 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express DGK_-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 6812)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6812)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: DGK_-NanoLuc Fusion Vector
FEATURES             Location/Qualifiers
     source          1..6812
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     672..692
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     972..991
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1033..1052
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1033..1051
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3291..3803
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     primer_bind     complement(3891..3909)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3946..4067
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3957..3976)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4011..4030
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4312..4669
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4312..4503
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4335..4355)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4520..4655
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4684..4731
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4763..5557
                     /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(4817..4836)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5427..5446
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5621..5669
                     /note="synthetic polyadenylation signal"
     rep_origin      5826..6371
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     6272..6291
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6488..6505
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat
      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc
      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta
     1021 aggctagagt attaatacga ctcactatag ggctagcgat cgccatggca aaggagaggg
     1081 gactgatcag cccatccgac ttcgcccagc tgcagaagta catggagtat tctaccaaga
     1141 aggtgagcga cgtgctgaag ctgtttgagg atggcgagat ggccaagtac gtgcagggcg
     1201 acgccatcgg ctatgagggc ttccagcagt ttctgaagat ctacctggag gtggataatg
     1261 tgcctcggca cctgtctctg gccctgttcc agagctttga gaccggccac tgcctgaacg
     1321 agaccaatgt gacaaaggac gtggtgtgcc tgaatgacgt gagctgttac ttctctctgc
     1381 tggagggcgg cagaccagag gataagctgg agttcacctt taagctgtat gacacagata
     1441 ggaacggcat cctggacagc tccgaggtgg ataagatcat cctgcagatg atgcgcgtgg
     1501 ccgagtacct ggactgggat gtgagcgagc tgcggcccat cctgcaggag atgatgaagg
     1561 agatcgacta tgatggatct ggaagcgtgt cccaggcaga gtgggtgcgg gcaggagcca
     1621 ccacagtgcc tctgctggtg ctgctgggcc tggagatgac cctgaaggac gatggccagc
     1681 acatgtggcg gccaaagaga tttcctaggc cagtgtattg caacctgtgc gagtctagca
     1741 tcggcctggg caagcagggc ctgtcctgca acctgtgcaa gtacaccgtg cacgaccagt
     1801 gcgccatgaa ggccctgcct tgtgaggtgt ccacatatgc caagtctcgc aaggacatcg
     1861 gcgtgcagag ccacgtgtgg gtgcggggag gatgcgagtc cggccgctgc gatcggtgtc
     1921 agaagaagat ccgcatctac cacagcctga ccggcctgca ctgcgtgtgg tgtcacctgg
     1981 agatccacga cgattgtctg caggcagtgg gacacgagtg cgactgtgga ctgctgcgcg
     2041 atcacatcct gcccccttcc tctatctatc caagcgtgct ggcatccgga cctgacagga
     2101 agaacagcaa gacctcccag aagacaatgg acgatctgaa tctgtctacc agcgaggccc
     2161 tgaggatcga tccagtgcct aacacacacc cactgctggt gttcgtgaat cccaagtccg
     2221 gcggcaagca gggccagaga gtgctgtgga agttccagta catcctgaac ccaaggcagg
     2281 tgtttaatct gctgaaggac ggccccgaga tcggcctgag actgtttaag gacgtgcctg
     2341 atagcaggat cctggtgtgc ggaggcgacg gaaccgtggg ctggatcctg gagacaatcg
     2401 ataaggccaa cctgcccgtg ctgccaccag tggccgtgct gcctctgggc accggcaatg
     2461 atctggcaag atgtctgaga tggggaggag gatacgaggg acagaacctg gccaagatcc
     2521 tgaaggacct ggagatgtcc aaggtggtgc acatggatag atggtctgtg gaagtgatcc
     2581 cacagcagac agaggagaag tccgacccag tgcccttcca gatcatcaac aattattttt
     2641 ccatcggcgt ggatgcctct atcgcccacc gcttccacat catgcgggag aagtaccccg
     2701 agaagtttaa ctctaggatg aagaataagc tgtggtattt cgagtttgcc acctccgagt
     2761 ctatcttcag cacatgcaag aagctggagg agtccctgac cgtggagatc tgtggcaagc
     2821 ccctggacct gtctaatctg agcctggagg gcatcgccgt gctgaacatc ccaagcatgc
     2881 acggcggctc caatctgtgg ggcgacacac ggagacccca cggcgatatc tacggcatca
     2941 accaggccct gggagcaacc gccaaagtga tcacagaccc tgatatcctg aagacctgcg
     3001 tgccagacct gtctgataag agactggagg tggtgggact ggagggagca atcgagatgg
     3061 gccagatcta tacaaagctg aagaacgcag gcaggcgcct ggcaaagtgc agcgagatca
     3121 ccttccacac cacaaagaca ctgcccatgc agatcgacgg cgagccttgg atgcagaccc
     3181 catgtacaat caagatcacc cacaagaacc agatgcctat gctgatgggc cctccaccca
     3241 ggtctacaaa tttctttggc tttctgagcg tttctctcgg ctcgagcggc gtcttcacac
     3301 tcgaagattt cgttggggac tggcgacaga cagccggcta caacctggac caagtccttg
     3361 aacagggagg tgtgtccagt ttgtttcaga atctcggggt gtccgtaact ccgatccaaa
     3421 ggattgtcct gagcggtgaa aatgggctga agatcgacat ccatgtcatc atcccgtatg
     3481 aaggtctgag cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg
     3541 tggatgatca tcactttaag gtgatcctgc actatggcac actggtaatc gacggggtta
     3601 cgccgaacat gatcgactat ttcggacggc cgtatgaagg catcgccgtg ttcgacggca
     3661 aaaagatcac tgtaacaggg accctgtgga acggcaacaa aattatcgac gagcgcctga
     3721 tcaaccccga cggctccctg ctgttccgag taaccatcaa cggagtgacc ggctggcggc
     3781 tgtgcgaacg cattctggcg taaggccgcg actctagagt cgacctgcag gcatgcaagc
     3841 tgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca
     3901 ataactagca taaccccttg gggcggccgc ttcgagcaga catgataaga tacattgatg
     3961 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4021 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     4081 gcattcattt tatgtttcag gttcaggggg agatgtggga ggttttttta agcaagtaaa
     4141 acctctacaa atgtggtaaa atcgaatttt aacaaaatat taacgcttac aatttcctga
     4201 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgc ggatctgcgc
     4261 agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct tctgaggcgg
     4321 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     4381 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     4441 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     4501 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4561 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     4621 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agcttaatta
     4681 actgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
     4741 aactaaaccc aggaggcaga tcatgattga acaagatgga ttgcacgcag gttctccggc
     4801 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4861 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4921 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     4981 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     5041 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     5101 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     5161 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     5221 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     5281 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     5341 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     5401 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5461 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5521 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5581 accgaccaag cgacgcccaa cctgccatca cgatggccgc aataaaatat ctttattttc
     5641 attacatctg tgtgttggtt ttttgtgtga atcgatagcg ataaggatcc gctcatgcga
     5701 tgataagctg tcaaacatga gaattacaac ttatatcgta tggggctgac ttcaggtgct
     5761 acatttgaag agataaattg cactgaaatc tagaaatatt ttatctgatt aataagatga
     5821 tcttcttgag atcgttttgg tctgcgcgta atctcttgct ctgaaaacga aaaaaccgcc
     5881 ttgcagggcg gtttttcgaa ggttctctga gctaccaact ctttgaaccg aggtaactgg
     5941 cttggaggag cgcagtcacc aaaacttgtc ctttcagttt agccttaacc ggcgcatgac
     6001 ttcaagacta actcctctaa atcaattacc agtggctgct gccagtggtg cttttgcatg
     6061 tctttccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cggactgaac
     6121 ggggggttcg tgcatacagt ccagcttgga gcgaactgcc tacccggaac tgagtgtcag
     6181 gcgtggaatg agacaaacgc ggccataaca gcggaatgac accggtaaac cgaaaggcag
     6241 gaacaggaga gcgcacgagg gagccgccag ggggaaacgc ctggtatctt tatagtcctg
     6301 tcgggtttcg ccaccactga tttgagcgtc agatttcgtg atgcttgtca ggggggcgga
     6361 gcctatggaa aaacggcttt gccgcggccc tctcacttcc ctgttaagta tcttcctggc
     6421 atcttccagg aaatctccgc cccgttcgta agccatttcc gctcgccgca gtcgaacgac
     6481 cgagcgtagc gagtcagtga gcgaggaagc ggaatatatc ctgtatcaca tattctgctg
     6541 acgcaccggt gcagcctttt ttctcctgcc acatgaagca cttcactgac accctcatca
     6601 gtgccaacat agtaagccag tatacactcc gctagcgctg atgtccggcg gtgcttttgc
     6661 cgttacgcac caccccgtca tgagattacg cgttttagcg gtgcgtacaa ttaagggatt
     6721 atggtaaatc cacttactgt ctgccctcgt agccatcgag ataaaccgca gtactccggc
     6781 cacgatgcgt ccggcgtaga ggatcgagat ct
//
