LOCUS       sequence-71780-e        7957 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Destination dual luciferase reporter plasmid for checking 
            effectiveness of shRNA. Compatible with standard AttL1-AttL2 Gateway
            Entry vectors..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 7957)
  AUTHORS   Geiling B, Vandal G, Posner AR, de Bruyns A, Dutchak KL, Garnett S, 
            Dankort D
  TITLE     A modular lentiviral and retroviral construction system to rapidly 
            generate vectors for gene expression and gene knockdown in vitro and
            in vivo.
  JOURNAL   PLoS One. 2013 Oct 11;8(10):e76279. doi: 
            10.1371/journal.pone.0076279.
  PUBMED    24146852
REFERENCE   2  (bases 1 to 7957)
  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: "PLoS One.";
            date: "2013-10-11"; volume: "8(10):e76279. doi"; pages: " 
            10.1371/journal.pone.0076279"
COMMENT     Sequence Label: pCheck2 Dest (R1-R2)   #BGV129
FEATURES             Location/Qualifiers
     source          1..7957
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        62..419
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        62..253
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(85..105)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      270..405
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          489..621
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     604..623
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        666..684
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      688..697
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             694..1629
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     protein_bind    1649..1773
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(1802..1807,1808..1825,1826..1832)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             1886..2566
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     primer_bind     complement(1952..1971)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2886..3191
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3070..3089
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3232..3356)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    3372..3420
                     /note="synthetic polyadenylation signal"
     promoter        3429..4180
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      4210..4219
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4216..5868
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /db_xref="GI:42718124"
                     /protein_id="AAS38485.1"
                     /translation="MADAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     RBS             5175..5183
                     /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)"
     polyA_signal    5912..6033
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5923..5942)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5977..5996
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        6166..6270
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6271..6339,6340..7131)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6489..6508)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7302..7890
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7791..7810
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct
       61 tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
      121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
      181 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
      241 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
      301 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
      361 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
      421 agcttgattc ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca
      481 ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
      541 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
      601 actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga
      661 gtacttaata cgactcacta taggctagcc accatggctt ccaaggtgta cgaccccgag
      721 caacgcaaac gcatgatcac tgggcctcag tggtgggctc gctgcaagca aatgaacgtg
      781 ctggactcct tcatcaacta ctatgattcc gagaagcacg ccgagaacgc cgtgattttt
      841 ctgcatggta acgctgcctc cagctacctg tggaggcacg tcgtgcctca catcgagccc
      901 gtggctagat gcatcatccc tgatctgatc ggaatgggta agtccggcaa gagcgggaat
      961 ggctcatatc gcctcctgga tcactacaag tacctcaccg cttggttcga gctgctgaac
     1021 cttccaaaga aaatcatctt tgtgggccac gactgggggg cttgtctggc ctttcactac
     1081 tcctacgagc accaagacaa gatcaaggcc atcgtccatg ctgagagtgt cgtggacgtg
     1141 atcgagtcct gggacgagtg gcctgacatc gaggaggata tcgccctgat caagagcgaa
     1201 gagggcgaga aaatggtgct tgagaataac ttcttcgtcg agaccatgct cccaagcaag
     1261 atcatgcgga aactggagcc tgaggagttc gctgcctacc tggagccatt caaggagaag
     1321 ggcgaggtta gacggcctac cctctcctgg cctcgcgaga tccctctcgt taagggaggc
     1381 aagcccgacg tcgtccagat tgtccgcaac tacaacgcct accttcgggc cagcgacgat
     1441 ctgcctaaga tgttcatcga gtccgaccct gggttctttt ccaacgctat tgtcgaggga
     1501 gctaagaagt tccctaacac cgagttcgtg aaggtgaagg gcctccactt cagccaggag
     1561 gacgctccag atgaaatggg taagtacatc aagagcttcg tggagcgcgt gctgaagaac
     1621 gagcagtaat tctaggcgat cgctcgagac aagtttgtac aaaaaagctg aacgagaaac
     1681 gtaaaatgat ataaatatca atatattaaa ttagattttg cataaaaaac agactacata
     1741 atactgtaaa acacaacata tccagtcact atggcggccg gccgcattag gcaccccagg
     1801 ctttacactt tatgcttccg gctcgtataa tgtgtggatt ttgagttagg atccgtcgag
     1861 attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga
     1921 tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac
     1981 ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa
     2041 gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga
     2101 attccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta
     2161 caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga
     2221 tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc
     2281 ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag
     2341 tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac
     2401 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
     2461 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
     2521 cgatgagtgg caggcggggc gtaatctaga ggatccggct tactaaaagc cagataacag
     2581 tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga
     2641 agtatgtcaa aaagaggtat gctatgaagc agcgtattac agtgacagtt gacagcgaca
     2701 gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat
     2761 gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcagaaaatc aggaagggat
     2821 ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga acaggggctg
     2881 gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg
     2941 atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg
     3001 cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg
     3061 aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag
     3121 aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct
     3181 ggggaatata aatgtcaggc tcccttatac acagccagtc tgcaggtcga ccatagtgac
     3241 tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat
     3301 atattgatat ttatatcatt ttacgtttct cgttcagctt tcttgtacaa agtggtgcgg
     3361 ccgctggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg
     3421 aggatctaaa tgagtcttcg gacctcgcgg gggccgctta agcggtggtt agggtttgtc
     3481 tgacgcgggg ggagggggaa ggaacgaaac actctcattc ggaggcggct cggggtttgg
     3541 tcttggtggc cacgggcacg cagaagagcg ccgcgatcct cttaagcacc cccccgccct
     3601 ccgtggaggc gggggtttgg tcggcgggtg gtaactggcg ggccgctgac tcgggcgggt
     3661 cgcgcgcccc agagtgtgac cttttcggtc tgctcgcaga cccccgggcg gcgccgccgc
     3721 ggcggcgacg ggctcgctgg gtcctaggct ccatggggac cgtatacgtg gacaggctct
     3781 ggagcatccg cacgactgcg gtgatattac cggagacctt ctgcgggacg agccgggtca
     3841 cgcggctgac gcggagcgtc cgttgggcga caaacaccag gacggggcac aggtacacta
     3901 tcttgtcacc cggaggcgcg agggactgca ggagcttcag ggagtggcgc agctgcttca
     3961 tccccgtggc ccgttgctcg cgtttgctgg cggtgtcccc ggaagaaata tatttgcatg
     4021 tctttagttc tatgatgaca caaaccccgc ccagcgtctt gtcattggcg aattcgaaca
     4081 cgcagatgca gtcggggcgg cgcggtccca ggtccacttc gcatattaag gtgacgcgtg
     4141 tggcctcgaa caccgagcga ccctgcagcg acccgcttaa aagcttggca ttccggtact
     4201 gttggtaaag ccaccatggc cgatgctaag aacattaaga agggccctgc tcccttctac
     4261 cctctggagg atggcaccgc tggcgagcag ctgcacaagg ccatgaagag gtatgccctg
     4321 gtgcctggca ccattgcctt caccgatgcc cacattgagg tggacatcac ctatgccgag
     4381 tacttcgaga tgtctgtgcg cctggccgag gccatgaaga ggtacggcct gaacaccaac
     4441 caccgcatcg tggtgtgctc tgagaactct ctgcagttct tcatgccagt gctgggcgcc
     4501 ctgttcatcg gagtggccgt ggcccctgct aacgacattt acaacgagcg cgagctgctg
     4561 aacagcatgg gcatttctca gcctaccgtg gtgttcgtgt ctaagaaggg cctgcagaag
     4621 atcctgaacg tgcagaagaa gctgcctatc atccagaaga tcatcatcat ggactctaag
     4681 accgactacc agggcttcca gagcatgtac acattcgtga catctcatct gcctcctggc
     4741 ttcaacgagt acgacttcgt gccagagtct ttcgacaggg acaaaaccat tgccctgatc
     4801 atgaacagct ctgggtctac cggcctgcct aagggcgtgg ccctgcctca tcgcaccgcc
     4861 tgtgtgcgct tctctcacgc ccgcgaccct attttcggca accagatcat ccccgacacc
     4921 gctattctga gcgtggtgcc attccaccac ggcttcggca tgttcaccac cctgggctac
     4981 ctgatttgcg gctttcgggt ggtgctgatg taccgcttcg aggaggagct gttcctgcgc
     5041 agcctgcaag actacaaaat tcagtctgcc ctgctggtgc caaccctgtt cagcttcttc
     5101 gctaagagca ccctgatcga caagtacgac ctgtctaacc tgcacgagat tgcctctggc
     5161 ggcgccccac tgtctaagga ggtgggcgaa gccgtggcca agcgctttca tctgccaggc
     5221 atccgccagg gctacggcct gaccgagaca accagcgcca ttctgattac cccagagggc
     5281 gacgacaagc ctggcgccgt gggcaaggtg gtgccattct tcgaggccaa ggtggtggac
     5341 ctggacaccg gcaagaccct gggagtgaac cagcgcggcg agctgtgtgt gcgcggccct
     5401 atgattatgt ccggctacgt gaataaccct gaggccacaa acgccctgat cgacaaggac
     5461 ggctggctgc actctggcga cattgcctac tgggacgagg acgagcactt cttcatcgtg
     5521 gaccgcctga agtctctgat caagtacaag ggctaccagg tggccccagc cgagctggag
     5581 tctatcctgc tgcagcaccc taacattttc gacgccggag tggccggcct gcccgacgac
     5641 gatgccggcg agctgcctgc cgccgtcgtc gtgctggaac acggcaagac catgaccgag
     5701 aaggagatcg tggactatgt ggccagccag gtgacaaccg ccaagaagct gcgcggcgga
     5761 gtggtgttcg tggacgaggt gcccaagggc ctgaccggca agctggacgc ccgcaagatc
     5821 cgcgagatcc tgatcaaggc taagaaaggc ggcaagatcg ccgtgtaata attctagagt
     5881 cggggcggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc
     5941 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     6001 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     6061 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     6121 ggtaaaatcg ataaggatcc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     6181 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     6241 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     6301 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     6361 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     6421 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     6481 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     6541 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     6601 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     6661 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     6721 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     6781 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     6841 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     6901 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     6961 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     7021 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     7081 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     7141 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     7201 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     7261 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     7321 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     7381 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     7441 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     7501 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     7561 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     7621 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     7681 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     7741 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     7801 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     7861 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     7921 ctggcctttt gctggccttt tgctcacatg gctcgac
//
