LOCUS       sequence-10551-e        4604 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows expression of shRNA from U6 promoter and a rescue cDNA from a
            doxycycline (Tet) inducible promoter in the same plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4604)
  AUTHORS   Ma HT, On KF, Tsang YH, Poon RY
  TITLE     An inducible system for expression and validation of the specificity
            of short hairpin RNA in mammalian cells.
  JOURNAL   Nucleic Acids Res. 2007  . 35(4):e22.
  PUBMED    17234679
REFERENCE   2  (bases 1 to 4604)
  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: "Nucleic 
            Acids Res. 2007  . 35(4):e22."
COMMENT     Sequence Label: pKAR1
FEATURES             Location/Qualifiers
     source          1..4604
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    join(15..33,34..56,57..75,76..98,99..117,118..140,141..159,
                     160..182,183..201,202..224,225..243,244..266,267..285)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        318..356
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     353..377
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             459..482
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             489..512
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     polyA_signal    544..678
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        1016..1327
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     1246..1266
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     CDS             join(1347..1349,1350..1352,1353..2063)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1347..1349,1350..1352,1353..2063)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1392..1413)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1653..1672)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2000..2021
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2108..2242
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2340..2360)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2340..2358)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2379..2395)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2379..2395)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2392..2414)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2403..2419
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2427..2433,2434..2451,2452..2457))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2472..2493
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2610..2627)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2781..3369)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2861..2880)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3540..4331,4332..4400))
                     /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     4163..4182
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4401..4505)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4573..4591
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt taccactccc
       61 tatcagtgat agagaaaagt gaaagtcgag tttaccactc cctatcagtg atagagaaaa
      121 gtgaaagtcg agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc
      181 actccctatc agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag
      241 agaaaagtga aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag
      301 ctcggtaccc gggtcgaggt aggcgtgtac ggtgggaggc ctatataagc agagctcgtt
      361 tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagag
      421 accgggaccg atccagcctc cgcgggctag ccacaatgga ctacaaagac gatgacgata
      481 aagccatgct ggaagttctg tttcaggggc ccatggctcg agaattcgag ctcggtaccc
      541 ggggatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
      601 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
      661 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
      721 gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat
      781 gatcctgcaa gcctcgtcgt cctggccgga ccacgctatc tgtgcaaggt ccccggcccc
      841 ggacgcgcgc tccatgagca gagcgcccgc cgccgaggcg aagcttcggg cggcgccctg
      901 cccgtcccac caggtcaaca ggcggtaacc ggcctcttca tcgggaatgc gcgcgacctt
      961 cagcatcgcc ggcatgtccc cctggcggac gggaagtatc agctcgacca agcttatccg
     1021 acgccgcatc tctaggcccg cgccggcccc ctcgcacaga cttgtgggag aagctcggct
     1081 actcccctgc cccggttaat ttgcatataa tatttcctag taactataga ggcttaatgt
     1141 gcgataaaag acagataatc tgttcttttt aatactagct acattttaca tgataggctt
     1201 ggatttctat aagagataca aatactaaat tattatttta aaaaacagca caaaaggaaa
     1261 ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat aaatatccct tggagaaaag
     1321 ccttgtttgt ggtcttcgga tccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     1381 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     1441 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     1501 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     1561 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     1621 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     1681 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     1741 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     1801 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     1861 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     1921 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     1981 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2041 tcggcatgga cgagctgtac aagggtggag ggaattcaag gcctctcgag cctctagata
     2101 atacgtagat ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     2161 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     2221 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     2281 gggggaggtg tgggaggttt tttaattctt gcggccgcgg cggtacccag cttttgttcc
     2341 ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga
     2401 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     2461 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     2521 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     2581 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2641 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2701 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2761 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2821 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2881 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2941 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt
     3001 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3061 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3121 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3181 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     3241 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3301 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3361 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3421 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3481 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3541 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3601 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3661 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3721 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3781 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3841 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3901 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3961 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4021 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     4081 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     4141 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     4201 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     4261 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     4321 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     4381 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4441 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4501 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     4561 ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttta
//
