LOCUS       Exported                7759 bp ds-DNA     circular SYN 07-JUN-2022
DEFINITION  Expresses a Cdkn2a-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    Lenti-sgCdkn2a#2/Cre
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 Jun 7, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7759
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        complement(20..124)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(199..218)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        390..769
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        771..969
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     924..944
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             987..1167
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1214..1339
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1836..2069
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2254..2298
                     /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    2596..2713
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2795..3035
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     2795..2815
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2966..2985
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3064..3139
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(3227..3250)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3624..3643
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3695..3715
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3716..4744
                     /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    4800..5388
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4853..4873)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5271..5282)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5391..5407)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5392..5408)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5917..6097
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6159..6747)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6239..6258)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6918..19)
                     /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     7541..7560
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
       61 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
      121 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
      181 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
      241 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
      301 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
      361 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
      421 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      481 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      541 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      601 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      661 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      721 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      781 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      841 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      901 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      961 tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc tgagcctggg
     1021 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     1081 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     1141 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
     1201 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
     1261 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
     1321 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
     1381 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
     1441 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
     1501 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
     1561 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     1621 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
     1681 cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta
     1741 tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga
     1801 agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg
     1861 ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa
     1921 ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag
     1981 catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg
     2041 gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt
     2101 tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg
     2161 aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac
     2221 tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta
     2281 gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa
     2341 ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct
     2401 atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc
     2461 ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag agacagagac
     2521 agatccattc gattagtgaa cggatcggca ctgcgtgcgc caattctgca gacaaatggc
     2581 agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag
     2641 aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa
     2701 aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttggt tagtaccggg
     2761 cccgctctag ccctgcagga actgtttaaa ctgtgagggc ctatttccca tgattccttc
     2821 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa
     2881 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc
     2941 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt
     3001 cgatttcttg gctttatata tcttgtggaa aggaccaaac accgcagctc ttctgctcaa
     3061 ctagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     3121 agtggcaccg agtcggtgct tttttgtttg gcgcgcctat ggcttaatta acatggcggc
     3181 cgcattctac cgggtagggg aggcgctttt cccaaggcag tctggagcat gcgctttagc
     3241 agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc
     3301 caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc
     3361 tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat
     3421 ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg
     3481 ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag
     3541 gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc
     3601 ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct
     3661 gttctcctct tcctcatctc cgggacccgg gatgcccaag aagaagagga aggtgtccaa
     3721 tttactgacc gtacaccaaa atttgcctgc attaccggtc gatgcaacga gtgatgaggt
     3781 tcgcaagaac ctgatggaca tgttcaggga tcgccaggcg ttttctgagc atacctggaa
     3841 aatgcttctg tccgtttgcc ggtcgtgggc ggcatggtgc aagttgaata accggaaatg
     3901 gtttcccgca gaacctgaag atgttcgcga ttatcttcta tatcttcagg cgcgcggtct
     3961 ggcagtaaaa actatccagc aacatttggg ccagctaaac atgcttcatc gtcggtccgg
     4021 gctgccacga ccaagtgaca gcaatgctgt ttcactggtt atgcggcgga tccgaaaaga
     4081 aaacgttgat gccggtgaac gtgcaaaaca ggctctagcg ttcgaacgca ctgatttcga
     4141 ccaggttcgt tcactcatgg aaaatagcga tcgctgccag gatatacgta atctggcatt
     4201 tctggggatt gcttataaca ccctgttacg tatagccgaa attgccagga tcagggttaa
     4261 agatatctca cgtactgacg gtgggagaat gttaatccat attggcagaa cgaaaacgct
     4321 ggttagcacc gcaggtgtag agaaggcact tagcctgggg gtaactaaac tggtcgagcg
     4381 atggatttcc gtctctggtg tagctgatga tccgaataac tacctgtttt gccgggtcag
     4441 aaaaaatggt gttgccgcgc catctgccac cagccagcta tcaactcgcg ccctggaagg
     4501 gatttttgaa gcaactcatc gattgattta cggcgctaag gatgactctg gtcagagata
     4561 cctggcctgg tctggacaca gtgcccgtgt cggagccgcg cgagatatgg cccgcgctgg
     4621 agtttcaata ccggagatca tgcaagctgg tggctggacc aatgtaaata ttgtcatgaa
     4681 ctatatccgt aacctggata gtgaaacagg ggcaatggtg cgcctgctgg aagatggcga
     4741 ttagccatgt ttaagggttc cggttccact aggtacaatt cgatatcaag cttatcgata
     4801 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     4861 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     4921 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     4981 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     5041 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     5101 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     5161 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     5221 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     5281 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     5341 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc
     5401 gacctcgatc gagacctaga aaaacatgga gcaatcacaa gtagcaatac agcagctacc
     5461 aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt tccagtcaca
     5521 cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     5581 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tatccttgat
     5641 ctgtggatct accacacaca aggctacttc cctgattggc agaactacac accagggcca
     5701 gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt tgagcaagag
     5761 aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt gagcctgcat
     5821 gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg cctagcattt
     5881 catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt agaccagatc
     5941 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     6001 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     6061 ctcagaccct tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa aggccagcaa
     6121 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6181 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6241 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6301 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6361 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6421 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6481 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6541 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6601 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6661 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6721 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6781 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6841 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6901 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6961 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     7021 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     7081 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7141 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7201 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7261 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7321 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7381 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7441 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7501 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7561 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7621 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7681 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7741 aagggaataa gggcgacac
//
