LOCUS       sequence-181198-        7645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet on shKras for conditional Kras knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7645)
  AUTHORS   Shao DD, Xue W, Krall EB, Bhutkar A, Piccioni F, Wang X, Schinzel 
            AC, Sood S, Rosenbluh J, Kim JW, Zwang Y, Roberts TM, Root DE, Jacks
            T, Hahn WC
  TITLE     KRAS and YAP1 Converge to Regulate EMT and Tumor Survival.
  JOURNAL   Cell. 2014 Jul 3;158(1):171-84. doi: 10.1016/j.cell.2014.06.004. 
            Epub 2014 Jun 19.
  PUBMED    24954536
REFERENCE   2  (bases 1 to 7645)
  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"; doi: 
            "10.1016/j.cell.2014.06.004"; journalName: "Cell"; date: "2014-07-3-
            3"; volume: "158"; issue: "1"; pages: "171-84"
COMMENT     Sequence Label: TGMP.shKras.1422
FEATURES             Location/Qualifiers
     source          1..7645
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     protein_bind    join(1451..1469,1470..1492,1493..1511,1512..1534,1535..1553,
                     1554..1576,1577..1595,1596..1618,1619..1637,1638..1660,
                     1661..1679,1680..1702,1703..1721)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        1754..1791
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1788..1812
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             join(1885..1887,1888..1890,1891..2604)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1885..1887,1888..1890,1891..2601)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1930..1951)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2191..2210)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2538..2559
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           2648..2769
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2797..2811
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           2839..2965
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     promoter        2979..3478
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3014..3037)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3411..3430
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3499..4098
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3499..3518)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3995..4015
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4690..4706)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4690..4706)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4703..4725)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4714..4730
                     /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(4738..4744,4745..4762,4763..4768))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4783..4804
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4921..4938)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5092..5680)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5172..5191)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5851..6642,6643..6711))
                     /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     6474..6493
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6712..6816)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6884..6902
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6940..6962)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7062..7081
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7275..7297
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7425..7444
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7644..7645,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatctggggg atcgatcctc tagagcatcg
     1441 agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc
     1501 agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga
     1561 aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc
     1621 cctatcagtg atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa
     1681 aagtgaaagt cgagtttacc actccctatc agtgatagag aaaagtgaaa gtcgagctcg
     1741 gtacccgggt cgagtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg
     1801 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg
     1861 gaccgatcca gcctctcgac caccatggtg agcaagggcg aggagctgtt caccggggtg
     1921 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1981 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     2041 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     2101 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     2161 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     2221 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     2281 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     2341 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     2401 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     2461 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     2521 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     2581 ggcatggacg agctgtacaa gtaatgacgt acggctaagc ctcgactagg gataacaggg
     2641 taattgtttg aatgaggctt cagtacttta cagaatcgtt gcctgcacat cttggaaaca
     2701 cttgctggga ttacttcttc aggttaaccc aacagaaggc tcgagaaggt atattgctgt
     2761 tgacagtgag cgacagaccc agtatgaaat agtatagtga agccacagat gtatactatt
     2821 tcatactggg tctgctgcct actgcctcgg aattcaaggg gctactttag gagcaattat
     2881 cttgtttact aaaactgaat accttgctat ctctttgata catttttaca aagctgaatt
     2941 aaaatggtat aaattaaatc acttttttca attctaccgg gtaggggagg cgcttttccc
     3001 aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg
     3061 cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg
     3121 tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca
     3181 gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat
     3241 ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct
     3301 ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag
     3361 gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac
     3421 gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac
     3481 ctgcagccca agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     3541 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     3601 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     3661 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     3721 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     3781 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     3841 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     3901 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     3961 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     4021 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     4081 cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca
     4141 cgaccccatg catcgataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaatg
     4201 aaagacccca cctgtaggtt tggcaactag agaaccatca gatgtttcca gggtgcccca
     4261 aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct
     4321 gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc actcggcgcg
     4381 ccagtcctcc gatagactgc gtcgcccggg tacccgtgta tccaataaac cctcttgcag
     4441 ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac
     4501 ccgtcagcgg gggtctttca tgggtaacag tttcttgaag ttggagaaca acattctgag
     4561 ggtaggagtc gaatattaag taatcctgac tcaattagcc actgttttga atccacatac
     4621 tccaatactc ctgaaatagt tcattatgga cagcgcagaa gagctgggga gaattaattc
     4681 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4741 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4801 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4861 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4921 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4981 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5041 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5101 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5161 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5221 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5281 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5341 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5401 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5461 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5521 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5581 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5641 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5701 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5761 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5821 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5881 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5941 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6001 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6061 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6121 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6181 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6241 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6301 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6361 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6421 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6481 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6541 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6601 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6661 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6721 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6781 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6841 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     6901 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     6961 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     7021 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     7081 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     7141 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     7201 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     7261 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gcgcaaggaa tggtgcatgc
     7321 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     7381 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     7441 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     7501 aggcgattag tccaatttgt taaagacagg atatcagtgg tccaggctct agttttgact
     7561 caacaatatc accagctgaa gcctatagag tacgagccat agataaaata aaagatttta
     7621 tttagtctcc agaaaaaggg gggaa
//
