LOCUS       sequence-471862-        5465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express hCHMP4B-NanoLuc(R) 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 5465)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5465)
  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: 
            "Unpublished"
COMMENT     Sequence Label: hCHMP4B-NanoLuc Vector
FEATURES             Location/Qualifiers
     source          1..5465
                     /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(1519..1537)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1520..1537)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     enhancer        1657..1960
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     2115..2135
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        2168..2215
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2247..3041
                     /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(2301..2320)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2911..2930
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(3199..3787)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3279..3298)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3913..3961
                     /note="synthetic polyadenylation signal"
     CDS             complement(3985..5022)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             3998..4006
                     /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(5053..5410)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5067..5202
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5219..5410
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5367..5387
                     /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 agagaaacca tggatccagc ccagttctcc aattccttca
      841 tgtcgtcgtc ctcctcttct ttcttcttgg cgggttttga tggtagggct atagagggaa
      901 catttggtag agggactgtt tcgggtccac tgatttccag caaattcttg tctagttcct
      961 cctgttctag ttcttctaat tccgccatga gctcatcctc gtcaaactct tctccaaacc
     1021 ctacaggttt cgaaattgct gttgaaatct cctctgcaag ttcttgctgg tcagcaatgt
     1081 cctgcattaa ctcatcaact ttatcgatgt ccatgttgtc atgggccgcc ttcatggcct
     1141 tggcggcata gcccatgttc ttgagcacct cggtgttggt gttggcattc tccagggcct
     1201 cccgctggaa ctcgatggtt gataatgtgc cgtcgatctg cgccagctgc ttctcatacc
     1261 tcttcttacg cttcagtgcc tggagggccg cgcgcttgtt tttggtgccg tgcttcttgg
     1321 cggccgtcag ctcctgctcg attttcttct ccaggaactc ctgtttcttg cttaacatct
     1381 cttccgtgtc ccgcagccgc tggatggcct cctggggggt cgggccgccc ttgccggcct
     1441 taccccctcc agccccgaac agcttcccga acaccgacat ggcgatcgct agctgcaaaa
     1501 agaacaagta gcttgtattc tatagtgtca cctaaatggg cgatctgacg gttcactaaa
     1561 ccagctctgc ttatataggc ctccctggcg cgccactagt tattaatagt aatcaattac
     1621 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
     1681 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     1741 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     1801 tgcccacttg gcagtacatc aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa
     1861 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg actttcctac
     1921 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     1981 caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     2041 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa
     2101 ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtca aatcgaattc
     2161 taatactgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt
     2221 gaggaactaa acccaggagg cagatcatga ttgaacaaga tggattgcac gcaggttctc
     2281 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     2341 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     2401 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     2461 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     2521 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     2581 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     2641 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     2701 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     2761 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     2821 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     2881 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     2941 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3001 agcgcatcgc cttctatcgc cttcttgacg agttcttctg attcgaaatg accgaccaag
     3061 cgacgcccaa ccggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3121 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3181 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3241 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3301 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3361 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3421 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3481 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3541 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3601 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     3661 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3721 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3781 atttcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3841 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttata
     3901 gtccggaatc gtcacacaaa aaaccaacac acagatgtaa tgaaaataaa gatattttat
     3961 tgcggccatc gtgatggcta gagtctaaac tcgacctacc tccttagcgc gcggtcgtgt
     4021 actgggccgg cggttgccgc tgtcggccag cacctcgacg cagccgtcgg tccatacggc
     4081 tgcgctccgg cgagcgattt gtgtgcgacc gacggtgccg gccccgctgc ggacgatggc
     4141 atcgcagcgg ccttgagccc aggcagcatc gtcgaagttg ccgtccacga ggctctggta
     4201 gagctgatcc aggccgatgc gcagcatgta ggctcgcaga cgagggctgc cggccagctc
     4261 gggatgccgg cgctcgaagt agcgagtctg ctgctccatg caagccagcc agggccgcca
     4321 gaagaagatg ttggccacct cgtactgact gtccccgaac atagcttcgg accagtcgat
     4381 tacggcggtg atgcggccgt tgtcggtcag gacgttgttg ctgccgaagt cggcatggac
     4441 gaggtggcgc acctcgggac agtcttcggc ccacagcatc agttcgtcca gggcttgagc
     4501 tacgctggcg gacacggtgt cgtccatcac ggtctgccag tggtagacat ggggatcagc
     4561 aatggcgcaa atgaaatccc gccaggttgt gtactggccg atgccttggg gcccgaatgg
     4621 gccgaagccg ctggtttgac tgaggtcggc ggcggcaata gcatccatag cttcggcgac
     4681 aggctgtaac acagcaggca gctctgtttc ggggaggtct tggagagtaa cgccttgggc
     4741 gcgtctactg atgcagtatg tcaggctctc gctgaactcg ccgatgtcca acacttcggg
     4801 gatgggtagt gcagcgctgg cgaagtggcg gtacacgtag cggtctttgt agaagccatc
     4861 agcgcagcta ttcacccgca gtacatagcc gcgtccgccg acatcgaagc tgaaggctcg
     4921 gctctcttcg ccctccgaca actgcatcag gtcgctcaca ctgtcgaact tctcgatgag
     4981 aaatttttca acgctggtag cggtgagttc gggcttcttc atggtggcgc tagtgtcaga
     5041 agaatcgagc tttttgcaaa agcctaggcc tccaaaaaag cctcctcact acttctggaa
     5101 tagctcagag gccgaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg
     5161 gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg
     5221 gactatggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     5281 tggggacttt ccacacctgg ttgctgacta attgagatgc atgctttgca tacttctgcc
     5341 tgctggggag cctggggact ttccacaccc taactgacac acattccaca gctggttctt
     5401 tccgcctcag aaggtaccta accaagttcc tctttcagag gttatttcag gccatggtgc
     5461 tgcgc
//
