LOCUS       sequence-363578-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Ncoa6-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7759)
  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 7759)
  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-sgNcoa6#1/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(91..110)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        282..661
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        663..861
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     816..836
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             879..1059
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1106..1231
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1728..1961
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2146..2190
                     /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    2488..2605
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2687..2935
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2687..2707
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2858..2877
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2956..3031
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3084..3583
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3119..3142)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3516..3535
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3587..3607
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3608..4636
                     /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    4692..5280
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4745..4765)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5163..5174)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5283..5299)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5284..5300)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5809..5989
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6051..6639)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6131..6150)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6810..7601,7602..7670))
                     /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     7433..7452
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7671..7759,1..16))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
       61 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
      121 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
      181 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
      241 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
      301 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      361 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      421 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      481 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      541 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      601 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      661 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      721 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      781 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      841 gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg ttagaccaga
      901 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
      961 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     1021 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
     1081 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
     1141 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
     1201 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
     1261 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     1321 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     1381 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     1441 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     1501 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     1561 acagcaagcg gccggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
     1621 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     1681 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     1741 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     1801 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     1861 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     1921 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     1981 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
     2041 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     2101 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     2161 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     2221 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     2281 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     2341 cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag
     2401 agagacagag acagatccat tcgattagtg aacggatcgg cactgcgtgc gccaattctg
     2461 cagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     2521 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     2581 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg
     2641 gttagtaccg ggcccgctct agccctgcag gaactgttta aactgtgagg gcctatttcc
     2701 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     2761 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     2821 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     2881 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggaccaa acaccgtgcc
     2941 caacttgtta cacatgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     3001 tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt tggcgcgcct atggcttaat
     3061 taacatggcg gccgcattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     3121 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     3181 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc
     3241 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     3301 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga
     3361 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     3421 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg
     3481 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
     3541 gtctgccgcg ctgttctcct cttcctcatc tccgggaccc gggatgccca agaagaagag
     3601 gaaggtgtcc aatttactga ccgtacacca aaatttgcct gcattaccgg tcgatgcaac
     3661 gagtgatgag gttcgcaaga acctgatgga catgttcagg gatcgccagg cgttttctga
     3721 gcatacctgg aaaatgcttc tgtccgtttg ccggtcgtgg gcggcatggt gcaagttgaa
     3781 taaccggaaa tggtttcccg cagaacctga agatgttcgc gattatcttc tatatcttca
     3841 ggcgcgcggt ctggcagtaa aaactatcca gcaacatttg ggccagctaa acatgcttca
     3901 tcgtcggtcc gggctgccac gaccaagtga cagcaatgct gtttcactgg ttatgcggcg
     3961 gatccgaaaa gaaaacgttg atgccggtga acgtgcaaaa caggctctag cgttcgaacg
     4021 cactgatttc gaccaggttc gttcactcat ggaaaatagc gatcgctgcc aggatatacg
     4081 taatctggca tttctgggga ttgcttataa caccctgtta cgtatagccg aaattgccag
     4141 gatcagggtt aaagatatct cacgtactga cggtgggaga atgttaatcc atattggcag
     4201 aacgaaaacg ctggttagca ccgcaggtgt agagaaggca cttagcctgg gggtaactaa
     4261 actggtcgag cgatggattt ccgtctctgg tgtagctgat gatccgaata actacctgtt
     4321 ttgccgggtc agaaaaaatg gtgttgccgc gccatctgcc accagccagc tatcaactcg
     4381 cgccctggaa gggatttttg aagcaactca tcgattgatt tacggcgcta aggatgactc
     4441 tggtcagaga tacctggcct ggtctggaca cagtgcccgt gtcggagccg cgcgagatat
     4501 ggcccgcgct ggagtttcaa taccggagat catgcaagct ggtggctgga ccaatgtaaa
     4561 tattgtcatg aactatatcc gtaacctgga tagtgaaaca ggggcaatgg tgcgcctgct
     4621 ggaagatggc gattagccat gtttaagggt tccggttcca ctaggtacaa ttcgatatca
     4681 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     4741 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     4801 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     4861 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     4921 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     4981 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     5041 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     5101 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     5161 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     5221 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     5281 atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac aagtagcaat
     5341 acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt
     5401 tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt
     5461 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     5521 gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac
     5581 acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca
     5641 gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct
     5701 gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc
     5761 cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg
     5821 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     5881 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     5941 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag catgtgagca
     6001 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6061 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6121 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6181 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6241 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6301 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6361 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6421 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6481 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6541 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6601 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6661 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6721 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6781 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6841 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6901 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6961 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7021 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7081 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7141 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7201 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7261 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7321 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7381 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7441 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7501 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7561 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7621 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7681 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7741 tgtatttaga aaaataaac
//
