LOCUS       sequence-180641-        7760 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses an Rb1-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7760)
  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 7760)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-sgRb1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /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        2897..2972
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3025..3524
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3060..3083)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3457..3476
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3528..3548
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3549..4577
                     /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    4633..5221
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4686..4706)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5104..5115)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5224..5240)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5225..5241)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5750..5930
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5992..6580)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6072..6091)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6751..7542,7543..7611))
                     /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     7374..7393
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7612..7716)
                     /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 acaccgtctt
     2881 accaggattc catccagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     2941 atcaacttga aaaagtggca ccgagtcggt gcttttttgt ttggcgcgcc tatggcttaa
     3001 ttaacatggc ggccgcattc taccgggtag gggaggcgct tttcccaagg cagtctggag
     3061 catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc tggcctcgca
     3121 cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc cccttcgcgc
     3181 caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca
     3241 ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac agcaccgctg
     3301 agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc tccttcgctt
     3361 tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc gggctcaggg
     3421 gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt caaaagcgca
     3481 cgtctgccgc gctgttctcc tcttcctcat ctccgggacc cgggatgccc aagaagaaga
     3541 ggaaggtgtc caatttactg accgtacacc aaaatttgcc tgcattaccg gtcgatgcaa
     3601 cgagtgatga ggttcgcaag aacctgatgg acatgttcag ggatcgccag gcgttttctg
     3661 agcatacctg gaaaatgctt ctgtccgttt gccggtcgtg ggcggcatgg tgcaagttga
     3721 ataaccggaa atggtttccc gcagaacctg aagatgttcg cgattatctt ctatatcttc
     3781 aggcgcgcgg tctggcagta aaaactatcc agcaacattt gggccagcta aacatgcttc
     3841 atcgtcggtc cgggctgcca cgaccaagtg acagcaatgc tgtttcactg gttatgcggc
     3901 ggatccgaaa agaaaacgtt gatgccggtg aacgtgcaaa acaggctcta gcgttcgaac
     3961 gcactgattt cgaccaggtt cgttcactca tggaaaatag cgatcgctgc caggatatac
     4021 gtaatctggc atttctgggg attgcttata acaccctgtt acgtatagcc gaaattgcca
     4081 ggatcagggt taaagatatc tcacgtactg acggtgggag aatgttaatc catattggca
     4141 gaacgaaaac gctggttagc accgcaggtg tagagaaggc acttagcctg ggggtaacta
     4201 aactggtcga gcgatggatt tccgtctctg gtgtagctga tgatccgaat aactacctgt
     4261 tttgccgggt cagaaaaaat ggtgttgccg cgccatctgc caccagccag ctatcaactc
     4321 gcgccctgga agggattttt gaagcaactc atcgattgat ttacggcgct aaggatgact
     4381 ctggtcagag atacctggcc tggtctggac acagtgcccg tgtcggagcc gcgcgagata
     4441 tggcccgcgc tggagtttca ataccggaga tcatgcaagc tggtggctgg accaatgtaa
     4501 atattgtcat gaactatatc cgtaacctgg atagtgaaac aggggcaatg gtgcgcctgc
     4561 tggaagatgg cgattagcca tgtttaaggg ttccggttcc actaggtaca attcgatatc
     4621 aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     4681 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     4741 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     4801 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     4861 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     4921 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     4981 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     5041 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     5101 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     5161 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     5221 catcgatacc gtcgacctcg atcgagacct agaaaaacat ggagcaatca caagtagcaa
     5281 tacagcagct accaatgctg attgtgcctg gctagaagca caagaggagg aggaggtggg
     5341 ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag ctgtagatct
     5401 tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc aacgaagaca
     5461 agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt ggcagaacta
     5521 cacaccaggg ccagggatca gatatccact gacctttgga tggtgctaca agctagtacc
     5581 agttgagcaa gagaaggtag aagaagccaa tgaaggagag aacacccgct tgttacaccc
     5641 tgtgagcctg catgggatgg atgacccgga gagagaagta ttagagtgga ggtttgacag
     5701 ccgcctagca tttcatcaca tggcccgaga gctgcatccg gactgtactg ggtctctctg
     5761 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     5821 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     5881 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gcatgtgagc
     5941 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     6001 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     6061 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     6121 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     6181 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     6241 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6301 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6361 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6421 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     6481 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     6541 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     6601 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6661 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6721 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6781 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6841 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6901 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6961 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     7021 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     7081 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     7141 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     7201 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     7261 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     7321 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     7381 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     7441 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     7501 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     7561 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     7621 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7681 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7741 tgacgtcgac ggatcgggag
//
