LOCUS       sequence-180661-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses an Rbm10-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7759)
  AUTHORS   Rogers ZN, McFarland CD, Winters IP, Naranjo S, Chuang CH, Petrov D,
            Winslow MM
  TITLE     A quantitative and multiplexed approach to uncover the fitness 
            landscape of tumor suppression in vivo.
  JOURNAL   Nat Methods. 2017 May 22. doi: 10.1038/nmeth.4297.
  PUBMED    28530655
REFERENCE   2  (bases 1 to 7759)
  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: "Nat 
            Methods. 2017 May 22. doi: 10.1038/nmeth.4297."
COMMENT     Sequence Label: Lenti-sgRbm10/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(31..50)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        222..601
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        603..801
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     756..776
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             819..999
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1046..1171
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1668..1901
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2086..2130
                     /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    2428..2545
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2627..2875
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2627..2647
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2798..2817
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2896..2971
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3024..3523
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3059..3082)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3456..3475
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3527..3547
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3548..4576
                     /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    4632..5220
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4685..4705)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5103..5114)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5223..5239)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5224..5240)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5749..5929
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5991..6579)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6071..6090)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6750..7541,7542..7610))
                     /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     7373..7392
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7611..7715)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
       61 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
      121 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
      181 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
      241 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      301 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      361 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      421 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      481 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      541 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      601 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      661 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      721 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      781 gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg ttagaccaga
      841 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
      901 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
      961 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
     1021 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
     1081 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
     1141 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
     1201 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     1261 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     1321 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     1381 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     1441 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     1501 acagcaagcg gccggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg
     1561 agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc
     1621 aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc
     1681 cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta
     1741 caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt
     1801 gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga
     1861 atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct
     1921 ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg
     1981 gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca
     2041 agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa
     2101 ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg
     2161 tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt
     2221 gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc
     2281 cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga aggtggagag
     2341 agagacagag acagatccat tcgattagtg aacggatcgg cactgcgtgc gccaattctg
     2401 cagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     2461 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     2521 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg
     2581 gttagtaccg ggcccgctct agccctgcag gaactgttta aactgtgagg gcctatttcc
     2641 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     2701 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     2761 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     2821 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggaccaa acaccgtatt
     2881 tcctgaacag atccggtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     2941 tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt tggcgcgcct atggcttaat
     3001 taacatggcg gccgcattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     3061 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     3121 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc
     3181 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     3241 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga
     3301 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     3361 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctaaggggcg ggctcagggg
     3421 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
     3481 gtctgccgcg ctgttctcct cttcctcatc tccgggaccc gggatgccca agaagaagag
     3541 gaaggtgtcc aatttactga ccgtacacca aaatttgcct gcattaccgg tcgatgcaac
     3601 gagtgatgag gttcgcaaga acctgatgga catgttcagg gatcgccagg cgttttctga
     3661 gcatacctgg aaaatgcttc tgtccgtttg ccggtcgtgg gcggcatggt gcaagttgaa
     3721 taaccggaaa tggtttcccg cagaacctga agatgttcgc gattatcttc tatatcttca
     3781 ggcgcgcggt ctggcagtaa aaactatcca gcaacatttg ggccagctaa acatgcttca
     3841 tcgtcggtcc gggctgccac gaccaagtga cagcaatgct gtttcactgg ttatgcggcg
     3901 gatccgaaaa gaaaacgttg atgccggtga acgtgcaaaa caggctctag cgttcgaacg
     3961 cactgatttc gaccaggttc gttcactcat ggaaaatagc gatcgctgcc aggatatacg
     4021 taatctggca tttctgggga ttgcttataa caccctgtta cgtatagccg aaattgccag
     4081 gatcagggtt aaagatatct cacgtactga cggtgggaga atgttaatcc atattggcag
     4141 aacgaaaacg ctggttagca ccgcaggtgt agagaaggca cttagcctgg gggtaactaa
     4201 actggtcgag cgatggattt ccgtctctgg tgtagctgat gatccgaata actacctgtt
     4261 ttgccgggtc agaaaaaatg gtgttgccgc gccatctgcc accagccagc tatcaactcg
     4321 cgccctggaa gggatttttg aagcaactca tcgattgatt tacggcgcta aggatgactc
     4381 tggtcagaga tacctggcct ggtctggaca cagtgcccgt gtcggagccg cgcgagatat
     4441 ggcccgcgct ggagtttcaa taccggagat catgcaagct ggtggctgga ccaatgtaaa
     4501 tattgtcatg aactatatcc gtaacctgga tagtgaaaca ggggcaatgg tgcgcctgct
     4561 ggaagatggc gattagccat gtttaagggt tccggttcca ctaggtacaa ttcgatatca
     4621 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     4681 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     4741 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     4801 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     4861 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     4921 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     4981 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     5041 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     5101 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     5161 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     5221 atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac aagtagcaat
     5281 acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt
     5341 tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt
     5401 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     5461 gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac
     5521 acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca
     5581 gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct
     5641 gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc
     5701 cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg
     5761 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     5821 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     5881 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag catgtgagca
     5941 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6001 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6061 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6121 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6181 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6241 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6301 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6361 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6421 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6481 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6541 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6601 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6661 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6721 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6781 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6841 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6901 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6961 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7021 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7081 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7141 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7201 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7261 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7321 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7381 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7441 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7501 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7561 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7621 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7681 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7741 gacgtcgacg gatcgggag
//
