LOCUS       sequence-361943-        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#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(136..155)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        327..706
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        708..906
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     861..881
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             924..1104
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1151..1276
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1773..2006
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2191..2235
                     /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    2533..2650
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2732..2980
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2732..2752
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2903..2922
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3002..3077
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3130..3629
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3165..3188)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3562..3581
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3633..3653
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3654..4682
                     /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    4738..5326
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4791..4811)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5209..5220)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5329..5345)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5330..5346)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5855..6035
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6097..6685)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6177..6196)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6856..7647,7648..7716))
                     /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     7479..7498
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7717..7760,1..61))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
       61 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
      121 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
      181 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
      241 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
      301 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
      361 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      421 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      481 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      541 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      601 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      661 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      721 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      781 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      841 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      901 aagcagcgcg ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc
      961 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     1021 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     1081 agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa
     1141 accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga
     1201 ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga
     1261 tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg
     1321 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
     1381 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
     1441 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
     1501 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
     1561 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgg
     1621 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
     1681 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
     1741 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
     1801 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
     1861 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
     1921 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
     1981 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
     2041 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
     2101 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
     2161 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
     2221 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
     2281 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
     2341 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     2401 aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga
     2461 tccattcgat tagtgaacgg atcggcactg cgtgcgccaa ttctgcagac aaatggcagt
     2521 attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat
     2581 agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat
     2641 tcaaaatttt cgggtttatt acagggacag cagagatcca gtttggttag taccgggccc
     2701 gctctagccc tgcaggaact gtttaaactg tgagggccta tttcccatga ttccttcata
     2761 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     2821 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     2881 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     2941 tttcttggct ttatatatct tgtggaaagg accaaacacc gccaaacgta aagcagcagt
     3001 ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     3061 tggcaccgag tcggtgcttt tttgtttggc gcgcctatgg cttaattaac atggcggccg
     3121 cattctaccg ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag
     3181 ccccgctggg cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca
     3241 ccggtaggcg ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc
     3301 ccctagtcag gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg
     3361 aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg
     3421 taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc
     3481 tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc
     3541 gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt
     3601 tctcctcttc ctcatctccg ggacccggga tgcccaagaa gaagaggaag gtgtccaatt
     3661 tactgaccgt acaccaaaat ttgcctgcat taccggtcga tgcaacgagt gatgaggttc
     3721 gcaagaacct gatggacatg ttcagggatc gccaggcgtt ttctgagcat acctggaaaa
     3781 tgcttctgtc cgtttgccgg tcgtgggcgg catggtgcaa gttgaataac cggaaatggt
     3841 ttcccgcaga acctgaagat gttcgcgatt atcttctata tcttcaggcg cgcggtctgg
     3901 cagtaaaaac tatccagcaa catttgggcc agctaaacat gcttcatcgt cggtccgggc
     3961 tgccacgacc aagtgacagc aatgctgttt cactggttat gcggcggatc cgaaaagaaa
     4021 acgttgatgc cggtgaacgt gcaaaacagg ctctagcgtt cgaacgcact gatttcgacc
     4081 aggttcgttc actcatggaa aatagcgatc gctgccagga tatacgtaat ctggcatttc
     4141 tggggattgc ttataacacc ctgttacgta tagccgaaat tgccaggatc agggttaaag
     4201 atatctcacg tactgacggt gggagaatgt taatccatat tggcagaacg aaaacgctgg
     4261 ttagcaccgc aggtgtagag aaggcactta gcctgggggt aactaaactg gtcgagcgat
     4321 ggatttccgt ctctggtgta gctgatgatc cgaataacta cctgttttgc cgggtcagaa
     4381 aaaatggtgt tgccgcgcca tctgccacca gccagctatc aactcgcgcc ctggaaggga
     4441 tttttgaagc aactcatcga ttgatttacg gcgctaagga tgactctggt cagagatacc
     4501 tggcctggtc tggacacagt gcccgtgtcg gagccgcgcg agatatggcc cgcgctggag
     4561 tttcaatacc ggagatcatg caagctggtg gctggaccaa tgtaaatatt gtcatgaact
     4621 atatccgtaa cctggatagt gaaacagggg caatggtgcg cctgctggaa gatggcgatt
     4681 agccatgttt aagggttccg gttccactag gtacaattcg atatcaagct tatcgataat
     4741 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     4801 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     4861 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     4921 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     4981 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     5041 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     5101 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     5161 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     5221 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     5281 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
     5341 cctcgatcga gacctagaaa aacatggagc aatcacaagt agcaatacag cagctaccaa
     5401 tgctgattgt gcctggctag aagcacaaga ggaggaggag gtgggttttc cagtcacacc
     5461 tcaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     5521 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagata tccttgatct
     5581 gtggatctac cacacacaag gctacttccc tgattggcag aactacacac cagggccagg
     5641 gatcagatat ccactgacct ttggatggtg ctacaagcta gtaccagttg agcaagagaa
     5701 ggtagaagaa gccaatgaag gagagaacac ccgcttgtta caccctgtga gcctgcatgg
     5761 gatggatgac ccggagagag aagtattaga gtggaggttt gacagccgcc tagcatttca
     5821 tcacatggcc cgagagctgc atccggactg tactgggtct ctctggttag accagatctg
     5881 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     5941 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     6001 cagacccttt tagtcagtgt ggaaaatctc tagcagcatg tgagcaaaag gccagcaaaa
     6061 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     6121 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     6181 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     6241 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6301 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6361 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6421 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6481 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6541 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6601 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6661 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6721 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6781 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6841 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6901 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6961 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     7021 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     7081 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     7141 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     7201 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     7261 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7321 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7381 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7441 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7501 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7561 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7621 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7681 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7741 aagcatttat cagggttatt
//
