LOCUS       sequence-359398-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Arhgap35-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-sgArhgap35#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(134..153)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        325..704
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        706..904
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     859..879
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             922..1102
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1149..1274
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1771..2004
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2189..2233
                     /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    2531..2648
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2730..2978
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2730..2750
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2901..2920
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3000..3075
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3128..3627
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3163..3186)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3560..3579
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3631..3651
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3652..4680
                     /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    4736..5324
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4789..4809)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5207..5218)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5327..5343)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5328..5344)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5853..6033
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6095..6683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6175..6194)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6854..7645,7646..7714))
                     /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     7477..7496
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7715..7760,1..59))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
       61 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat
      121 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
      181 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
      241 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
      301 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
      361 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      421 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      481 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      541 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      601 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      661 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      721 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      781 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      841 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      901 gcagcgcgtt ttgcctgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
      961 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     1021 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     1081 tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac
     1141 cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg
     1201 ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg
     1261 ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt
     1321 taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc
     1381 tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac
     1441 tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata
     1501 cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt
     1561 tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccggcc
     1621 gctgatcttc agacctggag gaggagatat gagggacaat tggagaagtg aattatataa
     1681 atataaagta gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt
     1741 ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc
     1801 agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt
     1861 gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct
     1921 gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag
     1981 atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac
     2041 cactgctgtg ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca
     2101 cacgacctgg atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt
     2161 aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa
     2221 atgggcaagt ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt
     2281 cataatgata gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt
     2341 gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag
     2401 gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc
     2461 cattcgatta gtgaacggat cggcactgcg tgcgccaatt ctgcagacaa atggcagtat
     2521 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag
     2581 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc
     2641 aaaattttcg ggtttattac agggacagca gagatccagt ttggttagta ccgggcccgc
     2701 tctagccctg caggaactgt ttaaactgtg agggcctatt tcccatgatt ccttcatatt
     2761 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     2821 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     2881 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     2941 tcttggcttt atatatcttg tggaaaggac caaacaccgc agaggtctct acaacctggg
     3001 ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg
     3061 gcaccgagtc ggtgcttttt tgtttggcgc gcctatggct taattaacat ggcggccgca
     3121 ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc
     3181 ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc
     3241 ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc
     3301 ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa
     3361 gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta
     3421 ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg
     3481 ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga
     3541 aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc
     3601 tcctcttcct catctccggg acccgggatg cccaagaaga agaggaaggt gtccaattta
     3661 ctgaccgtac accaaaattt gcctgcatta ccggtcgatg caacgagtga tgaggttcgc
     3721 aagaacctga tggacatgtt cagggatcgc caggcgtttt ctgagcatac ctggaaaatg
     3781 cttctgtccg tttgccggtc gtgggcggca tggtgcaagt tgaataaccg gaaatggttt
     3841 cccgcagaac ctgaagatgt tcgcgattat cttctatatc ttcaggcgcg cggtctggca
     3901 gtaaaaacta tccagcaaca tttgggccag ctaaacatgc ttcatcgtcg gtccgggctg
     3961 ccacgaccaa gtgacagcaa tgctgtttca ctggttatgc ggcggatccg aaaagaaaac
     4021 gttgatgccg gtgaacgtgc aaaacaggct ctagcgttcg aacgcactga tttcgaccag
     4081 gttcgttcac tcatggaaaa tagcgatcgc tgccaggata tacgtaatct ggcatttctg
     4141 gggattgctt ataacaccct gttacgtata gccgaaattg ccaggatcag ggttaaagat
     4201 atctcacgta ctgacggtgg gagaatgtta atccatattg gcagaacgaa aacgctggtt
     4261 agcaccgcag gtgtagagaa ggcacttagc ctgggggtaa ctaaactggt cgagcgatgg
     4321 atttccgtct ctggtgtagc tgatgatccg aataactacc tgttttgccg ggtcagaaaa
     4381 aatggtgttg ccgcgccatc tgccaccagc cagctatcaa ctcgcgccct ggaagggatt
     4441 tttgaagcaa ctcatcgatt gatttacggc gctaaggatg actctggtca gagatacctg
     4501 gcctggtctg gacacagtgc ccgtgtcgga gccgcgcgag atatggcccg cgctggagtt
     4561 tcaataccgg agatcatgca agctggtggc tggaccaatg taaatattgt catgaactat
     4621 atccgtaacc tggatagtga aacaggggca atggtgcgcc tgctggaaga tggcgattag
     4681 ccatgtttaa gggttccggt tccactaggt acaattcgat atcaagctta tcgataatca
     4741 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     4801 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     4861 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     4921 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     4981 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     5041 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     5101 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
     5161 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     5221 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     5281 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc
     5341 tcgatcgaga cctagaaaaa catggagcaa tcacaagtag caatacagca gctaccaatg
     5401 ctgattgtgc ctggctagaa gcacaagagg aggaggaggt gggttttcca gtcacacctc
     5461 aggtaccttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     5521 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatatc cttgatctgt
     5581 ggatctacca cacacaaggc tacttccctg attggcagaa ctacacacca gggccaggga
     5641 tcagatatcc actgaccttt ggatggtgct acaagctagt accagttgag caagagaagg
     5701 tagaagaagc caatgaagga gagaacaccc gcttgttaca ccctgtgagc ctgcatggga
     5761 tggatgaccc ggagagagaa gtattagagt ggaggtttga cagccgccta gcatttcatc
     5821 acatggcccg agagctgcat ccggactgta ctgggtctct ctggttagac cagatctgag
     5881 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     5941 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     6001 gaccctttta gtcagtgtgg aaaatctcta gcagcatgtg agcaaaaggc cagcaaaagg
     6061 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6121 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6181 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6241 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6301 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6361 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6421 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6481 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6541 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6601 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6661 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6721 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     6781 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     6841 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6901 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6961 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     7021 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     7081 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7141 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7201 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7261 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7321 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7381 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7441 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7501 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7561 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7621 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7681 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7741 gcatttatca gggttattgt
//
