LOCUS       sequence-363624-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Pten-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-sgPten#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(114..133)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        305..684
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        686..884
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     839..859
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             902..1082
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1129..1254
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1751..1984
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2169..2213
                     /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    2511..2628
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2710..2958
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2710..2730
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2881..2900
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2980..3055
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3108..3607
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3143..3166)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3540..3559
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3611..3631
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3632..4660
                     /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    4716..5304
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4769..4789)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5187..5198)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5307..5323)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5308..5324)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5833..6013
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6075..6663)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6155..6174)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6834..7625,7626..7694))
                     /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     7457..7476
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7695..7760,1..39))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
       61 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
      121 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
      181 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      241 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      301 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      361 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      421 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      481 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      541 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      601 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      661 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      721 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      781 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      841 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt ttgcctgtac
      901 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
      961 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     1021 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     1081 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
     1141 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
     1201 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
     1261 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
     1321 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
     1381 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     1441 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     1501 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     1561 caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc gctgatcttc agacctggag
     1621 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg
     1681 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag
     1741 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg
     1801 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc
     1861 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg
     1921 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc
     1981 tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg
     2041 ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg
     2101 acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc
     2161 agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt
     2221 ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct
     2281 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat
     2341 attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag
     2401 gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat
     2461 cggcactgcg tgcgccaatt ctgcagacaa atggcagtat tcatccacaa ttttaaaaga
     2521 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
     2581 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
     2641 agggacagca gagatccagt ttggttagta ccgggcccgc tctagccctg caggaactgt
     2701 ttaaactgtg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2761 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     2821 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     2881 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     2941 tggaaaggac caaacaccgt cacctggatt acagacccgg ttttagagct agaaatagca
     3001 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     3061 tgtttggcgc gcctatggct taattaacat ggcggccgca ttctaccggg taggggaggc
     3121 gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta
     3181 cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc
     3241 gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc
     3301 cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct
     3361 cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca
     3421 atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg
     3481 cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc
     3541 attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg
     3601 acccgggatg cccaagaaga agaggaaggt gtccaattta ctgaccgtac accaaaattt
     3661 gcctgcatta ccggtcgatg caacgagtga tgaggttcgc aagaacctga tggacatgtt
     3721 cagggatcgc caggcgtttt ctgagcatac ctggaaaatg cttctgtccg tttgccggtc
     3781 gtgggcggca tggtgcaagt tgaataaccg gaaatggttt cccgcagaac ctgaagatgt
     3841 tcgcgattat cttctatatc ttcaggcgcg cggtctggca gtaaaaacta tccagcaaca
     3901 tttgggccag ctaaacatgc ttcatcgtcg gtccgggctg ccacgaccaa gtgacagcaa
     3961 tgctgtttca ctggttatgc ggcggatccg aaaagaaaac gttgatgccg gtgaacgtgc
     4021 aaaacaggct ctagcgttcg aacgcactga tttcgaccag gttcgttcac tcatggaaaa
     4081 tagcgatcgc tgccaggata tacgtaatct ggcatttctg gggattgctt ataacaccct
     4141 gttacgtata gccgaaattg ccaggatcag ggttaaagat atctcacgta ctgacggtgg
     4201 gagaatgtta atccatattg gcagaacgaa aacgctggtt agcaccgcag gtgtagagaa
     4261 ggcacttagc ctgggggtaa ctaaactggt cgagcgatgg atttccgtct ctggtgtagc
     4321 tgatgatccg aataactacc tgttttgccg ggtcagaaaa aatggtgttg ccgcgccatc
     4381 tgccaccagc cagctatcaa ctcgcgccct ggaagggatt tttgaagcaa ctcatcgatt
     4441 gatttacggc gctaaggatg actctggtca gagatacctg gcctggtctg gacacagtgc
     4501 ccgtgtcgga gccgcgcgag atatggcccg cgctggagtt tcaataccgg agatcatgca
     4561 agctggtggc tggaccaatg taaatattgt catgaactat atccgtaacc tggatagtga
     4621 aacaggggca atggtgcgcc tgctggaaga tggcgattag ccatgtttaa gggttccggt
     4681 tccactaggt acaattcgat atcaagctta tcgataatca acctctggat tacaaaattt
     4741 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     4801 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     4861 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     4921 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     4981 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     5041 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     5101 tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc
     5161 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     5221 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     5281 tctccctttg ggccgcctcc ccgcatcgat accgtcgacc tcgatcgaga cctagaaaaa
     5341 catggagcaa tcacaagtag caatacagca gctaccaatg ctgattgtgc ctggctagaa
     5401 gcacaagagg aggaggaggt gggttttcca gtcacacctc aggtaccttt aagaccaatg
     5461 acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg
     5521 ctaattcact cccaacgaag acaagatatc cttgatctgt ggatctacca cacacaaggc
     5581 tacttccctg attggcagaa ctacacacca gggccaggga tcagatatcc actgaccttt
     5641 ggatggtgct acaagctagt accagttgag caagagaagg tagaagaagc caatgaagga
     5701 gagaacaccc gcttgttaca ccctgtgagc ctgcatggga tggatgaccc ggagagagaa
     5761 gtattagagt ggaggtttga cagccgccta gcatttcatc acatggcccg agagctgcat
     5821 ccggactgta ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     5881 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     5941 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     6001 aaaatctcta gcagcatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     6061 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     6121 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     6181 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     6241 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     6301 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6361 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6421 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6481 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6541 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6601 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6661 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6721 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6781 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6841 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6901 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6961 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     7021 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     7081 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     7141 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     7201 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     7261 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     7321 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7381 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7441 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7501 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7561 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7621 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7681 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7741 ctcatgagcg gatacatatt
//
