LOCUS       sequence-276273-        8244 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral tet-inducible expression of KrasG12D (mouse).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8244)
  AUTHORS   Su J, Morgani SM, David CJ, Wang Q, Er EE, Huang YH, Basnet H, Zou 
            Y, Shu W, Soni RK, Hendrickson RC, Hadjantonakis AK, Massague J
  TITLE     TGF-beta orchestrates fibrogenic and developmental EMTs via the RAS 
            effector RREB1.
  JOURNAL   Nature. 2020 Jan;577(7791):566-571. doi: 10.1038/s41586-019-1897-5. 
            Epub 2020 Jan 8.
  PUBMED    31915377
REFERENCE   2  (bases 1 to 8244)
  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: 
            "10.1038/s41586-019-1897-5"; journalName: "Nature"; date: "2020-01";
            volume: "577"; issue: "7791"; pages: "566-571"
COMMENT     Sequence Label: pLVX-Tight-Puro-KrasG12D2B
FEATURES             Location/Qualifiers
     source          1..8244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    134..268
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(158..177)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     212..231
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        316..420
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(421..489,490..1281)
                     /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(639..658)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1452..2040
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1941..1960
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2194..2211
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2328..2349
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2364..2369,2370..2387,2388..2394)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2402..2418
                     /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)."
     primer_bind     2407..2429
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2426..2442
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2426..2442
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     LTR             2570..3203
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    3410..3998
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3516..3527
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3925..3945
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4015..4614)
                     /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(4098..4118)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4595..4614
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(4635..5134)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4683..4702)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     5076..5099
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     promoter        complement(5725..6039)
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     primer_bind     complement(5728..5752)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    5799..5817
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5835..5853
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5870..5888
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5906..5924
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5942..5960
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    5977..5995
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6013..6031
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    6102..6217
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
     CDS             complement(6480..6524)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    6709..6942
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    7439..7564
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             7611..8244
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 aatttcttta aaatgaaaat aattcagagg aatcacaggt ttagagtaaa tgaaaccaca
       61 ggtaattggc agtggtaata gggtatgggg tgggaagttt gggatgattt tggttagctt
      121 gagttatcca gttgatccag acatgataag atacattgat gagtttggac aaaccacaac
      181 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
      241 aaccattata agctgcaata aacaagttga tctcccgatc cgtcgacgtc aggtggcact
      301 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
      361 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
      421 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
      481 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
      541 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
      601 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
      661 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
      721 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
      781 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
      841 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
      901 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
      961 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     1021 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     1081 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     1141 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     1201 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     1261 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     1321 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     1381 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     1441 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     1501 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     1561 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     1621 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1681 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1741 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1801 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     1861 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     1921 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     1981 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     2041 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     2101 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     2161 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     2221 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     2281 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     2341 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     2401 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctc
     2461 tagctagagg tcgacggtaa acgctagcaa tgtcaaggcc tctcactctc tgatattcat
     2521 ttctttgcaa gttataaata ctgaataata agatgacatg aactactact gctagagatt
     2581 ttccacactg actaaaaggg tctgagggat ctctagttac cagagtcaca caacagacgg
     2641 gcacacacta cttgaagcac tcaaggcaag ctttattgag gcttaagcag tgggttccct
     2701 agttagccag agagctccca ggctcagatc tggtctaacc agagagaccc agtacaggca
     2761 aaaagcagct gcttatatgc aggatctgag ggctcgccac tccccagtcc cgcccaggcc
     2821 acgcctccct ggaaagtccc cagcggaaag tcccttgtag caagctcgat atcagcagtt
     2881 cttgaagtac tccggatgca gctctcgggc cacgtgatga aatgctaggc ggctgtcaaa
     2941 cctccactct aacacttctc tctccgggtc atccatccca tgcaggctca cagggtgtaa
     3001 caagctggtg ttctctcctt tattggcctc ttctacctta tctggctcaa ctggtactag
     3061 cttgtagcac catccaaagg tcagtggata tctgacccct ggccctggtg tgtagttctg
     3121 ctaatcaggg aagtagcctt gtgtgtggta gatccacaga tcaaggatat cttgtcttcg
     3181 ttgggagtga attagccctt ccagtcccct cttttctttt aaaaagtggc taagatctac
     3241 agctgccttg taagtcattg gtcttaaagg tacctgaggt gtgactggaa aacccacctc
     3301 ctcctcctct tgtgcttcta gccaggcaca atcagcattg gtagctgctg tattgctact
     3361 tgtgattgct ccatgttttt ctaggtctcg actgcagaat taattccagg cggggaggcg
     3421 gcccaaaggg agatccgact cgtctgaggg cgaaggcgaa gacgcggaag aggccgcaga
     3481 gccggcagca ggccgcggga aggaaggtcc gctggattga gggccgaagg gacgtagcag
     3541 aaggacgtcc cgcgcagaat ccaggtggca acacaggcga gcagccatgg aaaggacgtc
     3601 agcttccccg acaacaccac ggaattgtca gtgcccaaca gccgagcccc tgtccagcag
     3661 cgggcaaggc aggcggcgat gagttccgcc gtggcaatag ggagggggaa agcgaaagtc
     3721 ccggaaagga gctgacaggt ggtggcaatg ccccaaccag tgggggttgc gtcagcaaac
     3781 acagtgcaca ccacgccacg ttgcctgaca acgggccaca actcctcata aagagacagc
     3841 aaccaggatt tatacaagga ggagaaaatg aaagccatac gggaagcaat agcatgatac
     3901 aaaggcatta aagcagcgta tccacatagc gtaaaaggag caacatagtt aagaatacca
     3961 gtcaatcttt cacaaatttt gtaatccaga ggttgattgt tccagacgcg cccgtcaggc
     4021 accgggcttg cgggtcatgc accaggtgcg cggtccttcg ggcacctcga cgtcggcggt
     4081 gacggtgaag ccgagccgct cgtagaaggg gaggttgcgg ggcgcggagg tctccaggaa
     4141 ggcgggcacc ccggcgcgct cggccgcctc cactccgggg agcacgacgg cgctgcccag
     4201 acccttgccc tggtggtcgg gcgagacgcc gacggtggcc aggaaccacg cgggctcctt
     4261 gggccggtgc ggcgccagga ggccttccat ctgttgctgc gcggccagcc gggaaccgct
     4321 caactcggcc atgcgcgggc cgatctcggc gaacaccgcc cccgcttcga cgctctccgg
     4381 cgtggtccag accgccaccg cggcgccgtc gtccgcgacc cacaccttgc cgatgtcgag
     4441 cccgacgcgc gtgaggaaga gttcttgcag ctcggtgacc cgctcgatgt ggcggtccgg
     4501 atcgacggtg tggcgcgtgg cggggtagtc ggcgaacgcg gcggcgaggg tgcgtacggc
     4561 cctggggacg tcgtcgcggg tggcgaggcg caccgtgggc ttgtactcgg tcatggtaag
     4621 cttgggctgc aggtcgaaag gcccggagat gaggaagagg agaacagcgc ggcagacgtg
     4681 cgcttttgaa gcgtgcagaa tgccgggcct ccggaggacc ttcgggcgcc cgccccgccc
     4741 ctgagcccgc ccctgagccc gcccccggac ccaccccttc ccagcctctg agcccagaaa
     4801 gcgaaggagc aaagctgcta ttggccgctg ccccaaaggc ctacccgctt ccattgctca
     4861 gcggtgctgt ccatctgcac gagactagtg agacgtgcta cttccatttg tcacgtcctg
     4921 cacgacgcga gctgcggggc gggggggaac ttcctgacta ggggaggagt agaaggtggc
     4981 gcgaaggggc caccaaagaa cggagccggt tggcgcctac cggtggatgt ggaatgtgtg
     5041 cgaggccaga ggccacttgt gtagcgccaa gtgcccagcg gggctgctaa agcgcatgct
     5101 ccagactgcc ttgggaaaag cgcctcccct acccggtaga attctcacat aactgtacac
     5161 cttgtccttg acttcttctt cttcttcttc ccatctttgc tcatcttttc tttatgtttt
     5221 cgaatttctc ggactaatgt atagaaggca tcgtcaacac cctgtcttgt ctttgctgag
     5281 gtctcaatga acggaatccc gtaactcctt gctaactcct gagcctgttt cgtgtctact
     5341 gttctagaag gcaaatcaca cttattccct accaggacca taggcacatc ttcagagtcc
     5401 tttactcttt taatttgttc tctataatgg tgaatatctt caaatgattt agtattattt
     5461 atggcaaata cacaaagaaa gccctcccca gttctcatgt actggtccct cattgcactg
     5521 tactcctctt gacctgctgt gtcgagaata tccaagagac aggtttctcc atcaattact
     5581 acttgtttcc tgtaggagtc ctctatcgta gggtcgtact catccacaaa gtgattctga
     5641 attagctgta tcgtcaaggc gctcttgcct acgccatcag ctccaaccac cacaagttta
     5701 tactcagtca tggatccttc tccaggcgat ctgacggttc actaaacgag ctctgcttat
     5761 ataggcctcc caccgtacac gcctacctcg acatacgttc tctatcactg atagggagta
     5821 aactcgacat acgttctcta tcactgatag ggataaactc gacatacgtt ctctatcact
     5881 gatagggagt aaactcgaca tacgttctct atcactgata gggagtaaac tcgacatacg
     5941 ttctctatca ctgataggga gtaaactcga catcgttctc tatcactgat agggagtaaa
     6001 ctcgacatac gttctctatc actgataggg agtaaactcg agtgaagacg aaagggcctc
     6061 atcgataaac tggatctctg ctgtccctgt aataaacccg aaaattttga atttttgtaa
     6121 tttgtttttg taattcttta gtttgtatgt ctgttgctat tatgtctact attctttccc
     6181 ctgcactgta ccccccaatc cccccttttc ttttaaaggc gataccgtcg agatccgttc
     6241 actaatcgaa tggatctgtc tctgtctctc tctccacctt cttcttctat tccttcgggc
     6301 ctgtcgggtc ccctcggggt tgggaggtgg gtctgaaacg ataatggtga atatccctgc
     6361 ctaactctat tcactataga aagtacagca aaaactattc ttaaacctac caagcctcct
     6421 actatcatta tgaataattt tatataccac agccaatttg ttatgttaaa ccaattccac
     6481 aaacttgccc atttatctaa ttccaataat tcttgttcat tcttttcttg ctggttttgc
     6541 gattcttcaa ttaaggagtg tattaagctt gtgtaattgt taatttctct gtcccactcc
     6601 atccaggtcg tgtgattcca aatctgttcc agagatttat tactccaact agcattccaa
     6661 ggcacagcag tggtgcaaat gagttttcca gagcaacccc aaatccccag gagctgttga
     6721 tcctttaggt atctttccac agccaggatt cttgcctgga gctgcttgat gccccagact
     6781 gtgagttgca acagatgctg ttgcgcctca atagccctca gcaaattgtt ctgctgctgc
     6841 actataccag acaataattg tctggcctgt accgtcagcg tcattgacgc tgcgcccata
     6901 gtgcttcctg ctgctcccaa gaacccaagg aacaaagctc ctattcccac tgctcttttt
     6961 tctctctgca ccactcttct ctttgccttg gtgggtgcta ctcctaatgg ttcaattttt
     7021 actactttat atttatataa ttcacttctc caattgtccc tcatatctcc tcctccaggt
     7081 ctgaagatca gcggccggcc gcttgctgtg cggtggtctt acttttgttt tgctcttcct
     7141 ctatcttgtc taaagcttcc ttggtgtctt ttatctctat cctttgatgc acacaataga
     7201 gggttgctac tgtattatat aatgatctaa gttcttctga tcctgtctga agggatggtt
     7261 gtagctgtcc cagtatttgt ctacagcctt ctgatgtttc taacaggcca ggattaactg
     7321 cgaatcgttc tagctccctg cttgcccata ctatatgttt taatttatat tttttctttc
     7381 cccctggcct taaccgaatt ttttcccatc gcgatctaat tctcccccgc ttaatactga
     7441 cgctctcgca cccatctctc tccttctagc ctccgctagt caaaattttt ggcgtactca
     7501 ccagtcgccg cccctcgcct cttgccgtgc gcgcttcagc aagccgagtc ctgcgtcgag
     7561 agagctcctc tggtttccct ttcgctttca agtccctgtt cgggcgccac tgctagagat
     7621 tttccacact gactaaaagg gtctgaggga tctctagtta ccagagtcac acaacagacg
     7681 ggcacacact acttgaagca ctcaaggcaa gctttattga ggcttaagca gtgggttccc
     7741 tagttagcca gagagctccc aggctcagat ctggtctaac cagagagacc cagtacaggc
     7801 aaaaagcagc tgcttatatg caggatctga gggctcgcca ctccccagtc ccgcccaggc
     7861 cacgcctccc tggaaagtcc ccagcggaaa gtcccttgta gcaagctcga tatcagcagt
     7921 tcttgaagta ctccggatgc agctctcggg ccacgtgatg aaatgctagg cggctgtcaa
     7981 acctccactc taacacttct ctctccgggt catccatccc atgcaggctc acagggtgta
     8041 acaagctggt gttctctcct ttattggcct cttctacctt atctggctca actggtacta
     8101 gcttgtagca ccatccaaag gtcagtggat atctgacccc tggccctggt gtgtagttct
     8161 gctaatcagg gaagtagcct tgtgtgtggt agatccacag atcaaggata tcttgtcttc
     8221 tttgggagtg aattagccct tcca
//
