LOCUS       sequence-359397-        7748 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 7748)
  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 7748)
  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#1/Cre
FEATURES             Location/Qualifiers
     source          1..7748
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(91..110)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        282..661
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        663..861
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     816..836
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             879..1059
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1106..1231
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1728..1961
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2146..2190
                     /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    2488..2605
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2687..2935
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2687..2707
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2858..2877
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2957..3032
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3085..3572
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3120..3143)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3505..3524
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3576..3596
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3597..4625
                     /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    4681..5269
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4734..4754)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5152..5163)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5272..5288)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5273..5289)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5798..5978
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6040..6628)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6120..6139)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6799..7590,7591..7659))
                     /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     7422..7441
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7660..7748,1..16))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
       61 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
      121 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
      181 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
      241 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
      301 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      361 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      421 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      481 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      541 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      601 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      661 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      721 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      781 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      841 gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg ttagaccaga
      901 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
      961 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     1021 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
     1081 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
     1141 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
     1201 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
     1261 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     1321 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     1381 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     1441 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     1501 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     1561 acagcaagcg gccggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
     1621 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     1681 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     1741 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     1801 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     1861 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     1921 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     1981 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
     2041 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     2101 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     2161 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     2221 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     2281 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     2341 cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag
     2401 agagacagag acagatccat tcgattagtg aacggatcgg cactgcgtgc gccaattctg
     2461 cagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     2521 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     2581 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg
     2641 gttagtaccg ggcccgctct agccctgcag gaactgttta aactgtgagg gcctatttcc
     2701 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     2761 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     2821 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     2881 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggaccaa acaccgagtg
     2941 tgatgagggc gttgaggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     3001 atcaacttga aaaagtggca ccgagtcggt gcttttttgt ttggcgcgcc tatggcttaa
     3061 ttaacatggc ggccgcattc taccgggtag gggaggcgct tttcccaagg cagtctggag
     3121 catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca
     3181 cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc
     3241 caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca
     3301 ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg
     3361 agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt
     3421 tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc ggggcgggcg
     3481 cccgaaggtc ctccggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc
     3541 tgttctcctc ttcctcatct ccgggacccg ggatgcccaa gaagaagagg aaggtgtcca
     3601 atttactgac cgtacaccaa aatttgcctg cattaccggt cgatgcaacg agtgatgagg
     3661 ttcgcaagaa cctgatggac atgttcaggg atcgccaggc gttttctgag catacctgga
     3721 aaatgcttct gtccgtttgc cggtcgtggg cggcatggtg caagttgaat aaccggaaat
     3781 ggtttcccgc agaacctgaa gatgttcgcg attatcttct atatcttcag gcgcgcggtc
     3841 tggcagtaaa aactatccag caacatttgg gccagctaaa catgcttcat cgtcggtccg
     3901 ggctgccacg accaagtgac agcaatgctg tttcactggt tatgcggcgg atccgaaaag
     3961 aaaacgttga tgccggtgaa cgtgcaaaac aggctctagc gttcgaacgc actgatttcg
     4021 accaggttcg ttcactcatg gaaaatagcg atcgctgcca ggatatacgt aatctggcat
     4081 ttctggggat tgcttataac accctgttac gtatagccga aattgccagg atcagggtta
     4141 aagatatctc acgtactgac ggtgggagaa tgttaatcca tattggcaga acgaaaacgc
     4201 tggttagcac cgcaggtgta gagaaggcac ttagcctggg ggtaactaaa ctggtcgagc
     4261 gatggatttc cgtctctggt gtagctgatg atccgaataa ctacctgttt tgccgggtca
     4321 gaaaaaatgg tgttgccgcg ccatctgcca ccagccagct atcaactcgc gccctggaag
     4381 ggatttttga agcaactcat cgattgattt acggcgctaa ggatgactct ggtcagagat
     4441 acctggcctg gtctggacac agtgcccgtg tcggagccgc gcgagatatg gcccgcgctg
     4501 gagtttcaat accggagatc atgcaagctg gtggctggac caatgtaaat attgtcatga
     4561 actatatccg taacctggat agtgaaacag gggcaatggt gcgcctgctg gaagatggcg
     4621 attagccatg tttaagggtt ccggttccac taggtacaat tcgatatcaa gcttatcgat
     4681 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     4741 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     4801 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     4861 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     4921 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     4981 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     5041 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     5101 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     5161 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     5221 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt
     5281 cgacctcgat cgagacctag aaaaacatgg agcaatcaca agtagcaata cagcagctac
     5341 caatgctgat tgtgcctggc tagaagcaca agaggaggag gaggtgggtt ttccagtcac
     5401 acctcaggta cctttaagac caatgactta caaggcagct gtagatctta gccacttttt
     5461 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atatccttga
     5521 tctgtggatc taccacacac aaggctactt ccctgattgg cagaactaca caccagggcc
     5581 agggatcaga tatccactga cctttggatg gtgctacaag ctagtaccag ttgagcaaga
     5641 gaaggtagaa gaagccaatg aaggagagaa cacccgcttg ttacaccctg tgagcctgca
     5701 tgggatggat gacccggaga gagaagtatt agagtggagg tttgacagcc gcctagcatt
     5761 tcatcacatg gcccgagagc tgcatccgga ctgtactggg tctctctggt tagaccagat
     5821 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     5881 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     5941 cctcagaccc ttttagtcag tgtggaaaat ctctagcagc atgtgagcaa aaggccagca
     6001 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     6061 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     6121 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6181 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6241 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6301 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6361 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6421 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6481 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6541 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6601 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6661 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6721 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6781 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6841 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6901 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6961 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     7021 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     7081 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     7141 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7201 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7261 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7321 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7381 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     7441 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7501 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7561 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7621 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7681 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7741 aaataaac
//
