LOCUS       sequence-479768-        6473 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express NanoLuc-AF9 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 6473)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6473)
  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            "Unpublished"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: NanoLuc-AF9 Vector
FEATURES             Location/Qualifiers
     source          1..6473
                     /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             1065..1577
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(3416..3434)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    3471..3592
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3482..3501)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3536..3555
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3837..4194
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3837..4028
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3860..3880)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4045..4180
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        4209..4256
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4288..5082
                     /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(4342..4361)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4952..4971
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5146..5194
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(5433..6021)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5513..5532)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    6137..6420
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
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 ggctagcgct caccatggtc ttcacactcg
     1081 aagatttcgt tggggactgg cgacagacag ccggctacaa cctggaccaa gtccttgaac
     1141 agggaggtgt gtccagtttg tttcagaatc tcggggtgtc cgtaactccg atccaaagga
     1201 ttgtcctgag cggtgaaaat gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag
     1261 gtctgagcgg cgaccaaatg ggccagatcg aaaaaatttt taaggtggtg taccctgtgg
     1321 atgatcatca ctttaaggtg atcctgcact atggcacact ggtaatcgac ggggttacgc
     1381 cgaacatgat cgactatttc ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa
     1441 agatcactgt aacagggacc ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca
     1501 accccgacgg ctccctgctg ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt
     1561 gcgaacgcat tctggcgggc tcgagcggcg cgatcgccat ggctagctcg tgtgccgtgc
     1621 aggtgaagct ggagctgggg caccgcgccc aggtgaggaa aaaacccacc gtggagggct
     1681 tcacccacga ctggatggtg ttcgtacgcg gtccggagca cagtaacata cagcactttg
     1741 tggagaaagt cgtcttccac ttgcacgaaa gctttcctag gccaaaaaga gtgtgcaaag
     1801 atccacctta caaagtagaa gaatctgggt atgctggttt cattttgcca attgaagttt
     1861 attttaaaaa caaggaagaa cctaggaaag tccgctttga ttatgactta ttcctgcatc
     1921 ttgaaggcca tccaccagtg aatcacctcc gctgtgaaaa gctaactttc aacaacccca
     1981 cagaggactt taggagaaag ttgctgaagg caggagggga ccctaatagg agtattcata
     2041 ccagcagcag cagcagcagc agcagtagca gcagcagcag cagcagcagc agcagcagta
     2101 gcagcagcag cagcagcagc agcagcagca gtagcagcag cagtagcagc agcagcagca
     2161 gcagtagtac cagtttttca aagcctcaca aattaatgaa ggagcacaag gaaaaacctt
     2221 ctaaagactc cagagaacat aaaagtgcct tcaaagaacc ttccagggat cacaacaaat
     2281 cttccaaaga atcctctaag aaacccaaag aaaataaacc actgaaagaa gagaaaatag
     2341 ttcctaagat ggccttcaag gaacctaaac ccatgtcaaa agagccaaaa ccagatagta
     2401 acttactcac catcaccagt ggacaagata agaaggctcc tagtaaaagg ccgcccattt
     2461 cagattctga agaactctca gccaaaaaaa ggaaaaagag tagctcagag gctttattta
     2521 aaagtttttc tagcgcacca ccactgatac tcacttgttc tgctgacaaa aaacagataa
     2581 aagataaatc tcatgtcaag atgggaaagg tcaaaattga aagtgagaca tcagagaaga
     2641 agaaatcaac gttaccgcca tttgatgata ttgtggatcc caatgattca gatgtggagg
     2701 agaatatatc ctctaaatct gattctgaac aacccagtcc tgccagctcc agctccagct
     2761 ccagctccag cttcacacca tcccagacca ggcaacaagg tcctttgagg tctataatga
     2821 aagatctgca ttctgatgac aatgaggagg aatcagatga agtggaggat aacgacaatg
     2881 actctgaaat ggagaggcct gtaaatagag gaggcagccg aagtcgcaga gttagcttaa
     2941 gtgatggcag cgatagtgaa agcagttctg cttcttcacc cctacatcac gaacctccac
     3001 cacccttact aaaaaccaac aacaaccaga ttcttgaagt gaaaagtcca ataaagcaaa
     3061 gcaaatcaga taagcaaata aagaatggtg aatgtgacaa ggcataccta gatgaactgg
     3121 tagagcttca cagaaggtta atgacattga gagaaagaca cattctgcag cagatcgtga
     3181 accttataga agaaactgga cactttcata tcacaaacac aacatttgat tttgatcttt
     3241 gctcgctgga caaaaccaca gtccgtaaac tacagagtta cctggaaaca tctggaacat
     3301 ccgtttaaac gaattcgggc tcggtacccg gggatcctct agagtcgacc tgcaggcatg
     3361 caagctgatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
     3421 gagcaataac tagcataacc ccttggggcg gccgcttcga gcagacatga taagatacat
     3481 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     3541 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     3601 caattgcatt cattttatgt ttcaggttca gggggagatg tgggaggttt ttttaagcaa
     3661 gtaaaacctc tacaaatgtg gtaaaatcga attttaacaa aatattaacg cttacaattt
     3721 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgcggatc
     3781 tgcgcagcac catggcctga aataacctct gaaagaggaa cttggttagg taccttctga
     3841 ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3901 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3961 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4021 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     4081 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     4141 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt
     4201 aattaactgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg
     4261 tgaggaacta aacccaggag gcagatcatg attgaacaag atggattgca cgcaggttct
     4321 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4381 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4441 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4501 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4561 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4621 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4681 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4741 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4801 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4861 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4921 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4981 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     5041 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     5101 aaatgaccga ccaagcgacg cccaacctgc catcacgatg gccgcaataa aatatcttta
     5161 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagcgataag gatcctcttt
     5221 gcgcttgcgt tttcccttgt ccagatagcc cagtagctga cattcatccg gggtcagcac
     5281 cgtttctgcg gactggcttt ctacccggta tcagctcact caaaggcggt aatacggtta
     5341 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5401 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5461 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5521 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5581 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5641 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5701 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5761 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5821 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
     5881 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5941 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6001 cgcagaaaaa aaggatttca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6061 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6121 tagatccttt tatagtccgg aaatacagga acgcacgctg gatggccctt cgctgggatg
     6181 gtgaaaccat gaaaaatggc agcttcagtg gattaagtgg gggtaatgtg gcctgtaccc
     6241 tctggttgca taggtattca tacggttaaa atttatcagg cgcgattgcg gcagtttttc
     6301 gggtggtttg ttgccatttt tacctgtctg ctgccgtgat cgcgctgaac gcgttttagc
     6361 ggtgcgtaca attaagggat tatggtaaat ccacttactg tctgccctcg tagccatcga
     6421 gataaaccgc agtactccgg ccacgatgcg tccggcgtag aggatcgaga tct
//
