LOCUS       sequence-363645-        7748 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Cmtr2-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7748)
  AUTHORS   Cai H, Chew SK, Li C, Tsai MK, Andrejka L, Murray CW, Hughes NW, 
            Shuldiner EG, Ashkin EL, Tang R, Hung KL, Chen LC, Lee SYC, Yousefi 
            M, Lin WY, Kunder CA, Cong L, McFarland CD, Petrov DA, Swanton C, 
            Winslow MM
  TITLE     A functional taxonomy of tumor suppression in oncogenic KRAS-driven 
            lung cancer.
  JOURNAL   Cancer Discov. 2021 Feb 19. pii: 2159-8290.CD-20-1325. doi: 
            10.1158/2159-8290.CD-20-1325.
  PUBMED    33608386
REFERENCE   2  (bases 1 to 7748)
  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: "Cancer 
            Discov. 2021 Feb 19. pii: 2159-8290.CD-20-1325. doi: 
            10.1158/2159-8290.CD-20-1325."
COMMENT     Sequence Label: Lenti-sgCmtr2#1/Cre
FEATURES             Location/Qualifiers
     source          1..7748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(121..140)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        312..691
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        693..891
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     846..866
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             909..1089
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1136..1261
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1758..1991
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2176..2220
                     /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    2518..2635
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2717..2965
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2717..2737
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2888..2907
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2987..3062
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3115..3602
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3150..3173)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3535..3554
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3606..3626
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3627..4655
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     misc_feature    4711..5299
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4764..4784)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5182..5193)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5302..5318)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5303..5319)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5828..6008
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6070..6658)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6150..6169)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6829..7620,7621..7689))
                     /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     7452..7471
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7690..7748,1..46))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
       61 aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag
      121 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
      181 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
      241 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
      301 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
      361 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
      421 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
      481 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
      541 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
      601 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
      661 catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
      721 gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg
      781 gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
      841 attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg
      901 cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
      961 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     1021 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     1081 tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc
     1141 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt
     1201 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt
     1261 cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg
     1321 aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc
     1381 agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca
     1441 accatccctt cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
     1501 ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag acaagataga
     1561 ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccggccgct gatcttcaga
     1621 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
     1681 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
     1741 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
     1801 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
     1861 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
     1921 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
     1981 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
     2041 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
     2101 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
     2161 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg
     2221 tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta
     2281 ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg
     2341 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg
     2401 cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg
     2461 aacggatcgg cactgcgtgc gccaattctg cagacaaatg gcagtattca tccacaattt
     2521 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     2581 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     2641 ttattacagg gacagcagag atccagtttg gttagtaccg ggcccgctct agccctgcag
     2701 gaactgttta aactgtgagg gcctatttcc catgattcct tcatatttgc atatacgata
     2761 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     2821 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     2881 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     2941 tatcttgtgg aaaggaccaa acaccgccca caggagaagc tctccggttt tagagctaga
     3001 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     3061 gcttttttgt ttggcgcgcc tatggcttaa ttaacatggc ggccgcattc taccgggtag
     3121 gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg ctgggcactt
     3181 ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt aggcgccaac
     3241 cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta gtcaggaagt
     3301 tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca
     3361 ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc ctttggggca
     3421 gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga aggggtgggt
     3481 ccgggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct
     3541 gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggacccg
     3601 ggatgcccaa gaagaagagg aaggtgtcca atttactgac cgtacaccaa aatttgcctg
     3661 cattaccggt cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac atgttcaggg
     3721 atcgccaggc gttttctgag catacctgga aaatgcttct gtccgtttgc cggtcgtggg
     3781 cggcatggtg caagttgaat aaccggaaat ggtttcccgc agaacctgaa gatgttcgcg
     3841 attatcttct atatcttcag gcgcgcggtc tggcagtaaa aactatccag caacatttgg
     3901 gccagctaaa catgcttcat cgtcggtccg ggctgccacg accaagtgac agcaatgctg
     3961 tttcactggt tatgcggcgg atccgaaaag aaaacgttga tgccggtgaa cgtgcaaaac
     4021 aggctctagc gttcgaacgc actgatttcg accaggttcg ttcactcatg gaaaatagcg
     4081 atcgctgcca ggatatacgt aatctggcat ttctggggat tgcttataac accctgttac
     4141 gtatagccga aattgccagg atcagggtta aagatatctc acgtactgac ggtgggagaa
     4201 tgttaatcca tattggcaga acgaaaacgc tggttagcac cgcaggtgta gagaaggcac
     4261 ttagcctggg ggtaactaaa ctggtcgagc gatggatttc cgtctctggt gtagctgatg
     4321 atccgaataa ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca
     4381 ccagccagct atcaactcgc gccctggaag ggatttttga agcaactcat cgattgattt
     4441 acggcgctaa ggatgactct ggtcagagat acctggcctg gtctggacac agtgcccgtg
     4501 tcggagccgc gcgagatatg gcccgcgctg gagtttcaat accggagatc atgcaagctg
     4561 gtggctggac caatgtaaat attgtcatga actatatccg taacctggat agtgaaacag
     4621 gggcaatggt gcgcctgctg gaagatggcg attagccatg tttaagggtt ccggttccac
     4681 taggtacaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     4741 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     4801 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     4861 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     4921 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     4981 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     5041 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     5101 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     5161 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     5221 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     5281 ctttgggccg cctccccgca tcgataccgt cgacctcgat cgagacctag aaaaacatgg
     5341 agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc tagaagcaca
     5401 agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac caatgactta
     5461 caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat
     5521 tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac aaggctactt
     5581 ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga cctttggatg
     5641 gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa
     5701 cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtatt
     5761 agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc tgcatccgga
     5821 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     5881 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     5941 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     6001 ctctagcagc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     6061 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     6121 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6181 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6241 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6301 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6361 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6421 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6481 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6541 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6601 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6661 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6721 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6781 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6841 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6901 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6961 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     7021 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     7081 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     7141 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7201 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     7261 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7321 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7381 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     7441 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     7501 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7561 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7621 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7681 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7741 gagcggat
//
