LOCUS       sequence-363584-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Nf1-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7760)
  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 7760)
  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-sgNf1#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(152..171)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        343..722
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        724..922
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     877..897
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             940..1120
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1167..1292
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1789..2022
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2207..2251
                     /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    2549..2666
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2748..2996
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2748..2768
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2919..2938
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3018..3093
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3146..3645
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3181..3204)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3578..3597
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3649..3669
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3670..4698
                     /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    4754..5342
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4807..4827)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5225..5236)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5345..5361)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5346..5362)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5871..6051
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6113..6701)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6193..6212)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6872..7663,7664..7732))
                     /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     7495..7514
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7733..7760,1..77))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
       61 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
      121 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
      181 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
      241 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
      301 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
      361 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
      421 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      481 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      541 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      601 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      661 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      721 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      781 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      841 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      901 ggtgggaggt ctatataagc agcgcgtttt gcctgtactg ggtctctctg gttagaccag
      961 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     1021 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     1081 tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg aacagggact
     1141 tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt gctgaagcgc
     1201 gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg actagcggag
     1261 gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga attagatcgc
     1321 gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta aaacatatag
     1381 tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta gaaacatcag
     1441 aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga tcagaagaac
     1501 ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg atagagataa
     1561 aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt aagaccaccg
     1621 cacagcaagc ggccggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     1681 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     1741 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     1801 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     1861 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     1921 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     1981 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     2041 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     2101 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     2161 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     2221 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     2281 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     2341 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     2401 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
     2461 gagagacaga gacagatcca ttcgattagt gaacggatcg gcactgcgtg cgccaattct
     2521 gcagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca
     2581 gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa
     2641 aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga gatccagttt
     2701 ggttagtacc gggcccgctc tagccctgca ggaactgttt aaactgtgag ggcctatttc
     2761 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     2821 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     2881 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     2941 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacca aacaccgctt
     3001 caacaactcc ctcgatggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     3061 tatcaacttg aaaaagtggc accgagtcgg tgcttttttg tttggcgcgc ctatggctta
     3121 attaacatgg cggccgcatt ctaccgggta ggggaggcgc ttttcccaag gcagtctgga
     3181 gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc
     3241 acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg
     3301 ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc
     3361 aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct
     3421 gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct
     3481 ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg
     3541 ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc
     3601 acgtctgccg cgctgttctc ctcttcctca tctccgggac ccgggatgcc caagaagaag
     3661 aggaaggtgt ccaatttact gaccgtacac caaaatttgc ctgcattacc ggtcgatgca
     3721 acgagtgatg aggttcgcaa gaacctgatg gacatgttca gggatcgcca ggcgttttct
     3781 gagcatacct ggaaaatgct tctgtccgtt tgccggtcgt gggcggcatg gtgcaagttg
     3841 aataaccgga aatggtttcc cgcagaacct gaagatgttc gcgattatct tctatatctt
     3901 caggcgcgcg gtctggcagt aaaaactatc cagcaacatt tgggccagct aaacatgctt
     3961 catcgtcggt ccgggctgcc acgaccaagt gacagcaatg ctgtttcact ggttatgcgg
     4021 cggatccgaa aagaaaacgt tgatgccggt gaacgtgcaa aacaggctct agcgttcgaa
     4081 cgcactgatt tcgaccaggt tcgttcactc atggaaaata gcgatcgctg ccaggatata
     4141 cgtaatctgg catttctggg gattgcttat aacaccctgt tacgtatagc cgaaattgcc
     4201 aggatcaggg ttaaagatat ctcacgtact gacggtggga gaatgttaat ccatattggc
     4261 agaacgaaaa cgctggttag caccgcaggt gtagagaagg cacttagcct gggggtaact
     4321 aaactggtcg agcgatggat ttccgtctct ggtgtagctg atgatccgaa taactacctg
     4381 ttttgccggg tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca gctatcaact
     4441 cgcgccctgg aagggatttt tgaagcaact catcgattga tttacggcgc taaggatgac
     4501 tctggtcaga gatacctggc ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat
     4561 atggcccgcg ctggagtttc aataccggag atcatgcaag ctggtggctg gaccaatgta
     4621 aatattgtca tgaactatat ccgtaacctg gatagtgaaa caggggcaat ggtgcgcctg
     4681 ctggaagatg gcgattagcc atgtttaagg gttccggttc cactaggtac aattcgatat
     4741 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     4801 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     4861 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     4921 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     4981 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     5041 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     5101 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     5161 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     5221 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     5281 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     5341 gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc acaagtagca
     5401 atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag gaggaggtgg
     5461 gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     5521 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     5581 aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat tggcagaact
     5641 acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac aagctagtac
     5701 cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc ttgttacacc
     5761 ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg aggtttgaca
     5821 gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact gggtctctct
     5881 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     5941 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     6001 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agcatgtgag
     6061 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6121 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6181 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6241 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6301 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6361 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6421 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6481 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6541 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6601 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6661 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6721 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6781 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6841 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6901 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6961 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     7021 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7081 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7141 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7201 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7261 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7321 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7381 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7441 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7501 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7561 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7621 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7681 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7741 tttttcaata ttattgaagc
//
