LOCUS       sequence-482015-        7447 bp    DNA     circular SYN 05-AUG-2025
DEFINITION  Express MPL-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 7447)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7447)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: MPL-NanoLuc Fusion Vector
FEATURES             Location/Qualifiers
     source          1..7447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     313..333
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        366..413
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             445..1104
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(511..530)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      complement(1276..1864)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1356..1375)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    1990..2038
                     /note="synthetic polyadenylation signal"
     CDS             complement(2062..2856)
                     /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(2173..2192)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2783..2802
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3234..4412)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3243..4181
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(3266..3286)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     polyA_signal    4429..4510
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4454..4473
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4687..5199)
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
                     VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
                     PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
                     LCERILA"
     promoter        complement(7164..7182)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(7165..7182)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        join(7302..7447,1..158)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg
       61 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt
      121 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
      181 ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
      241 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaataacc
      301 ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtcaaa tcgaattcta
      361 atactgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga
      421 ggaactaaac ccaggaggca gatcatggag aaaaaaatca ctggatatac caccgttgat
      481 atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc
      541 tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag
      601 cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa
      661 ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac
      721 accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat
      781 ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc
      841 tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt
      901 ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcact
      961 atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat
     1021 catgccgtct gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc
     1081 gatgagtggc agggcggggc gtaagcggga ctctggggtt cgaaatgacc gaccaagcga
     1141 cgcccaaccg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     1201 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     1261 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     1321 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     1381 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     1441 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     1501 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     1561 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     1621 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     1681 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     1741 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1801 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatt
     1861 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     1921 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttatagtc
     1981 cggaatcgtc acacaaaaaa ccaacacaca gatgtaatga aaataaagat attttattgc
     2041 ggccatcgtg atggctagag ttcagaagaa ctcgtcaaga aggcgataga aggcgatgcg
     2101 ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc
     2161 aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc
     2221 cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa
     2281 gcaggcatcg ccgtgggtca cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct
     2341 ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac
     2401 aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa
     2461 tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac
     2521 tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag
     2581 cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt
     2641 cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg caccggacag
     2701 gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc
     2761 agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc
     2821 cggagaacct gcgtgcaatc catcttgttc aatcatggtg gcatcgattc ccggtcaaca
     2881 tccagtacac ctccggcaca ccggcttccc caagggcgcc acgctctccc accacaacat
     2941 cctcaacaac ggctactggg tgggccgcac ggtcggctac acggagcagg accgggtgtg
     3001 tctgccggtg cccttctacc actgcttcgg catggtcatg ggcaacttgg gcgccacctc
     3061 ccacgccgcc tgcatcgtca tcccggcccc gtccttcgag ccggcggcga cgctggaggc
     3121 ggtgcagcgg gagaggtgca cgtccctgta cggcgtcccc acgaagccga attccaccac
     3181 actggactag atgcatgctc gacagtgtga tggatatctg cagaattcgg ctttcacgac
     3241 acctgaaatg gaagaaaaaa actttgaacc actgtctgag gcttgagaat gaaccaagat
     3301 ccaaactcaa aaagggcaaa ttccaaggag aattacatca agtgccaagc tggcctaact
     3361 tcagtctcca cccactcagt gtggggaaac tccatcgcat aaaacccctc cccccaacct
     3421 aaagacgacg tactccaaaa gctcgagaac taatcgaggt gcctggacgg cgcccggtac
     3481 tccgtggagt cacatgaagc gacggctgag gacggaaagg cccttttcct ttgtgtgggt
     3541 gactcacccg cccgctctcc cgagcgccgc gtcctccatt ttgagctgcc tgcagcaggg
     3601 ccgggaagcg gccatctttc cgctcacgca actggtgccg accgggccag ccttgccgcc
     3661 cagggcgggg cgatacacgg cggcgcgagg ccaggcacca gagcaggccg gccagcttga
     3721 gactaccccc gtccgattct cggtggccgc gctcgcaggc cccgcctcgc cgaacatgtg
     3781 cgctgggacg cacgggcccc gtcgccgccc gcggccccaa aaaccgaaat accagtgtgc
     3841 agatcttggc ccgcatttac aagactatct tgccagaaaa aaagcgtcgc agcaggtcat
     3901 caaaaatttt aaatggctag agacttatcg aaagcagcga gacaggcgcg aaggtgccac
     3961 cagattcgca cgcggcggcc ccagcgccca ggccaggcct caactcaagc acgaggcgaa
     4021 ggggctcctt aagcgcaagg cctcgaactc tcccacccac ttccaacccg aagctcggga
     4081 tcaagaatca cgtactgcag ccaggtggaa gtaattcaag gcacgcaagg gccataaccc
     4141 gtaaagaggc caggcccgcg ggaaccacac acggcactta cctgtgttct ggcggcaaac
     4201 ccgttgcgaa aaagaacgtt cacggcgact actgcactta tatacggttc tcccccaccc
     4261 tcgggaaaaa ggcggagcca gtacacgaca tcactttccc agtttacccc gcgccacctt
     4321 ctctaggcac cggttcaatt gccgacccct ccccccaact tctcggggac tgtgggcgat
     4381 gtgcgctctg cccactgacg ggcaccggag ccaagacgcg tgacgtcggc agtgaaaaaa
     4441 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     4501 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     4561 ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt atgatccgcg
     4621 gccgcaatac gtcgacgtta tcagctgact tcgtacgaga gcctaggtgt ttctagagtc
     4681 gcggccttac gccagaatgc gttcgcacag ccgccagccg gtcactccgt tgatggttac
     4741 tcggaacagc agggagccgt cggggttgat caggcgctcg tcgataattt tgttgccgtt
     4801 ccacagggtc cctgttacag tgatcttttt gccgtcgaac acggcgatgc cttcatacgg
     4861 ccgtccgaaa tagtcgatca tgttcggcgt aaccccgtcg attaccagtg tgccatagtg
     4921 caggatcacc ttaaagtgat gatcatccac agggtacacc accttaaaaa ttttttcgat
     4981 ctggcccatt tggtcgccgc tcagaccttc atacgggatg atgacatgga tgtcgatctt
     5041 cagcccattt tcaccgctca ggacaatcct ttggatcgga gttacggaca ccccgagatt
     5101 ctgaaacaaa ctggacacac ctccctgttc aaggacttgg tccaggttgt agccggctgt
     5161 ctgtcgccag tccccaacga aatcttcgag tgtgaagacg ccgctcgagc cgagagaaac
     5221 aggctgctgc caatagctta gtggtaggta ggaatggttg gcaatgtggg tggtacagca
     5281 ggaccctgac tcagccatgg gtgggcacac agacaggggc atggtcccca ggcaagaagg
     5341 ctgcaatctt cggtagtcca tctgggcctg ggaggaacac aggggcaaag gagtcctctc
     5401 tgaggacttg gggaggattt caaggaggct gggttccact tcttcacagg tatctgagac
     5461 tgtggccttg ggcgggctca gggctgcagt gtccctaagg tactggccta ggacccggtg
     5521 caggtctgga agtgagggcc acagggcatg cctcagtctc ctgtagtgtg caggaaactg
     5581 ccacctcagc agcagcaggc ccaggacggc gctgaggccc agcactagat gcagagcggt
     5641 caccaaggag atccaggcgg tctcggtggc ggtctccacc ctagttgggt ccgaccacga
     5701 gctccaggga ccttggtagg tggggccgtt gagcctggcg cgcagctgta aacggtagcg
     5761 agatcgcggg cgcagctcca gggtccctcc tcgggccccg agaggcggct ccagcacctt
     5821 ccagtcctga tggccttctc ctgtgtatcg gagttgataa caggtctctt gggctgccca
     5881 ggacgatggg tgctgccact ccaattccag atgcccactg gagatctccc tccagtgcaa
     5941 gtttggggtg gggaggcgca cagcctggtg gatccagaaa ggggagccca ggtagctgtg
     6001 aacagtaccc ggggctgtgg tcacctccac aaggatgtga ataatgctgt catttcgtga
     6061 cttgaagtgg cagcgagaga actgtggggt ctgtagtcct ggatttgttt tctcttcctc
     6121 ttcgcagttc tcccagatgg ggtacctgtc tctggggcag caccgtgccc tgctgtggta
     6181 gaagaagcct tgggagctag catggtcctg ttgctgccat tgacaggtaa cattcttcag
     6241 gtccaaggta aagcattgca gtccaagtgc cactgcatct ccaggcaggt ccacagtcac
     6301 agggagggac caggatcccc aggagccacc gagggagatc ccatcaggtt cgctgcgcag
     6361 ctgcagccag taggagttgc caggctggag tcctgagatg aggcagcttc caccctctgc
     6421 tgtcagagct gaagcttctc tacttgggga ggtctgcttt ggtccatctt gccagggcat
     6481 tgtgggctga gcacatggag actggtccag agcagaggct gagtgaggcc tctgcagagc
     6541 agggcagcag gtttctgtgg caatcagctg tatgaccgtg ggaccagtgg agttcttggg
     6601 atctctgggg ccatagcgga gttcgtacct caggaaatca ctgatttctg gagctggctc
     6661 ctcccagctg atctgaagtt cccctggctg gctcccaccc atggccttga tgatactggg
     6721 gggagccggc aggcctacac tgtccacaaa gaggactcgc tgagtccgag tctggtttag
     6781 gaacacattc ttcacccaga ggtgcagcgg aaagaagaga cgcacttcct cctggtctgg
     6841 aaactggcac acgtatcggg ttccaaagtg gggcatgctc tgggaactca gggggcaagc
     6901 acggggcttc tcccgcgggt aggcatacag cagctggtat gtcccactgg gcgctgcctc
     6961 ttcctcatcc cagaagcaag tgaggtcctc aaatgttcgg gagaaacact tcaggggctc
     7021 tgagtctgat gccagcaagg agacatcttg gctgctgact tgggccaggt tttgaggggc
     7081 caggaggagg caggaggtga ccatgaagag ggcccaggag ggcatggcga tcgctagctg
     7141 caaaaagaac aagtagcttg tattctatag tgtcacctaa atgggcgatc tgacggttca
     7201 ctaaaccagc tctgcttata taggcctccc tggcgcgcca ctagttatta atagtaatca
     7261 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     7321 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     7381 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     7441 taaactg
//
