LOCUS       sequence-363581-        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#2/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(64..83)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        255..634
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        636..834
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     789..809
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             852..1032
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1079..1204
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1701..1934
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2119..2163
                     /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    2461..2578
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2660..2908
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2660..2680
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2831..2850
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2929..3004
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3057..3556
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3092..3115)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3489..3508
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3560..3580
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3581..4609
                     /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    4665..5253
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4718..4738)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5136..5147)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5256..5272)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5257..5273)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5782..5962
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6024..6612)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6104..6123)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6783..7574,7575..7643))
                     /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     7406..7425
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7644..7748)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
       61 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
      121 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
      181 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
      241 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      301 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      361 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      421 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      481 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      541 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      601 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      661 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      721 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      781 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt
      841 ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
      901 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
      961 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     1021 aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct
     1081 ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact
     1141 ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag
     1201 cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg
     1261 gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt
     1321 cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct
     1381 acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac
     1441 cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat
     1501 agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc gctgatcttc
     1561 agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta
     1621 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga
     1681 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc
     1741 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata
     1801 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc
     1861 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag
     1921 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg
     1981 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg
     2041 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
     2101 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
     2161 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
     2221 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
     2281 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac
     2341 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta
     2401 gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa
     2461 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     2521 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
     2581 ggtttattac agggacagca gagatccagt ttggttagta ccgggcccgc tctagccctg
     2641 caggaactgt ttaaactgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg
     2701 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
     2761 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
     2821 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
     2881 atatatcttg tggaaaggac caaacaccga tgacatgttc ccgccctggt tttagagcta
     2941 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     3001 gtgctttttt gtttggcgcg cctatggctt aattaacatg gcggccgcat tctaccgggt
     3061 aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac
     3121 ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca
     3181 accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa
     3241 gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct
     3301 cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg
     3361 cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg
     3421 gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg
     3481 aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc
     3541 atctccggga cccgggatgc ccaagaagaa gaggaaggtg tccaatttac tgaccgtaca
     3601 ccaaaatttg cctgcattac cggtcgatgc aacgagtgat gaggttcgca agaacctgat
     3661 ggacatgttc agggatcgcc aggcgttttc tgagcatacc tggaaaatgc ttctgtccgt
     3721 ttgccggtcg tgggcggcat ggtgcaagtt gaataaccgg aaatggtttc ccgcagaacc
     3781 tgaagatgtt cgcgattatc ttctatatct tcaggcgcgc ggtctggcag taaaaactat
     3841 ccagcaacat ttgggccagc taaacatgct tcatcgtcgg tccgggctgc cacgaccaag
     3901 tgacagcaat gctgtttcac tggttatgcg gcggatccga aaagaaaacg ttgatgccgg
     3961 tgaacgtgca aaacaggctc tagcgttcga acgcactgat ttcgaccagg ttcgttcact
     4021 catggaaaat agcgatcgct gccaggatat acgtaatctg gcatttctgg ggattgctta
     4081 taacaccctg ttacgtatag ccgaaattgc caggatcagg gttaaagata tctcacgtac
     4141 tgacggtggg agaatgttaa tccatattgg cagaacgaaa acgctggtta gcaccgcagg
     4201 tgtagagaag gcacttagcc tgggggtaac taaactggtc gagcgatgga tttccgtctc
     4261 tggtgtagct gatgatccga ataactacct gttttgccgg gtcagaaaaa atggtgttgc
     4321 cgcgccatct gccaccagcc agctatcaac tcgcgccctg gaagggattt ttgaagcaac
     4381 tcatcgattg atttacggcg ctaaggatga ctctggtcag agatacctgg cctggtctgg
     4441 acacagtgcc cgtgtcggag ccgcgcgaga tatggcccgc gctggagttt caataccgga
     4501 gatcatgcaa gctggtggct ggaccaatgt aaatattgtc atgaactata tccgtaacct
     4561 ggatagtgaa acaggggcaa tggtgcgcct gctggaagat ggcgattagc catgtttaag
     4621 ggttccggtt ccactaggta caattcgata tcaagcttat cgataatcaa cctctggatt
     4681 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
     4741 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
     4801 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
     4861 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
     4921 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
     4981 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
     5041 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
     5101 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
     5161 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
     5221 cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct cgatcgagac
     5281 ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc tgattgtgcc
     5341 tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta
     5401 agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga
     5461 ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac
     5521 acacaaggct acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca
     5581 ctgacctttg gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc
     5641 aatgaaggag agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg
     5701 gagagagaag tattagagtg gaggtttgac agccgcctag catttcatca catggcccga
     5761 gagctgcatc cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     5821 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     5881 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     5941 tcagtgtgga aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6001 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6061 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6121 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6181 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6241 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6301 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6361 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6421 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6481 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6541 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6601 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6661 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6721 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6781 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6841 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6901 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6961 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     7021 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     7081 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7141 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7201 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7261 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7321 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7381 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7441 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7501 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7561 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7621 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7681 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7741 gttccgcgca catttcccc
//
