LOCUS       sequence-180659-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a non-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 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-sgNT3/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..58)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        230..609
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        611..809
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     764..784
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             827..1007
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1054..1179
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1676..1909
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2094..2138
                     /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    2436..2553
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2635..2883
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2635..2655
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2806..2825
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2905..2980
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3033..3532
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3068..3091)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3465..3484
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3536..3556
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3557..4585
                     /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    4641..5229
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4694..4714)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5112..5123)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5232..5248)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5233..5249)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5758..5938
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6000..6588)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6080..6099)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6759..7550,7551..7619))
                     /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     7382..7401
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7620..7724)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
       61 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
      121 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
      181 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      241 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      301 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      361 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      421 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      481 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      541 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      601 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      661 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      721 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      781 cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt
      841 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
      901 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
      961 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
     1021 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
     1081 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
     1141 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
     1201 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
     1261 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
     1321 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
     1381 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
     1441 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
     1501 accaccgcac agcaagcggc cggccgctga tcttcagacc tggaggagga gatatgaggg
     1561 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1621 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1681 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     1741 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1801 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1861 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     1921 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     1981 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     2041 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     2101 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     2161 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     2221 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     2281 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
     2341 gtggagagag agacagagac agatccattc gattagtgaa cggatcggca ctgcgtgcgc
     2401 caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
     2461 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     2521 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
     2581 ccagtttggt tagtaccggg cccgctctag ccctgcagga actgtttaaa ctgtgagggc
     2641 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     2701 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     2761 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     2821 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggaccaaac
     2881 accgccgcgc cgttagggaa cgaggtttta gagctagaaa tagcaagtta aaataaggct
     2941 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt ggcgcgccta
     3001 tggcttaatt aacatggcgg ccgcattcta ccgggtaggg gaggcgcttt tcccaaggca
     3061 gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg
     3121 gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc
     3181 cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc
     3241 gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag
     3301 caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc
     3361 cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg
     3421 gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca
     3481 aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggacccg ggatgcccaa
     3541 gaagaagagg aaggtgtcca atttactgac cgtacaccaa aatttgcctg cattaccggt
     3601 cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac atgttcaggg atcgccaggc
     3661 gttttctgag catacctgga aaatgcttct gtccgtttgc cggtcgtggg cggcatggtg
     3721 caagttgaat aaccggaaat ggtttcccgc agaacctgaa gatgttcgcg attatcttct
     3781 atatcttcag gcgcgcggtc tggcagtaaa aactatccag caacatttgg gccagctaaa
     3841 catgcttcat cgtcggtccg ggctgccacg accaagtgac agcaatgctg tttcactggt
     3901 tatgcggcgg atccgaaaag aaaacgttga tgccggtgaa cgtgcaaaac aggctctagc
     3961 gttcgaacgc actgatttcg accaggttcg ttcactcatg gaaaatagcg atcgctgcca
     4021 ggatatacgt aatctggcat ttctggggat tgcttataac accctgttac gtatagccga
     4081 aattgccagg atcagggtta aagatatctc acgtactgac ggtgggagaa tgttaatcca
     4141 tattggcaga acgaaaacgc tggttagcac cgcaggtgta gagaaggcac ttagcctggg
     4201 ggtaactaaa ctggtcgagc gatggatttc cgtctctggt gtagctgatg atccgaataa
     4261 ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca ccagccagct
     4321 atcaactcgc gccctggaag ggatttttga agcaactcat cgattgattt acggcgctaa
     4381 ggatgactct ggtcagagat acctggcctg gtctggacac agtgcccgtg tcggagccgc
     4441 gcgagatatg gcccgcgctg gagtttcaat accggagatc atgcaagctg gtggctggac
     4501 caatgtaaat attgtcatga actatatccg taacctggat agtgaaacag gggcaatggt
     4561 gcgcctgctg gaagatggcg attagccatg tttaagggtt ccggttccac taggtacaat
     4621 tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     4681 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     4741 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     4801 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     4861 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     4921 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     4981 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     5041 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     5101 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     5161 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     5221 cctccccgca tcgataccgt cgacctcgat cgagacctag aaaaacatgg agcaatcaca
     5281 agtagcaata cagcagctac caatgctgat tgtgcctggc tagaagcaca agaggaggag
     5341 gaggtgggtt ttccagtcac acctcaggta cctttaagac caatgactta caaggcagct
     5401 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     5461 cgaagacaag atatccttga tctgtggatc taccacacac aaggctactt ccctgattgg
     5521 cagaactaca caccagggcc agggatcaga tatccactga cctttggatg gtgctacaag
     5581 ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa cacccgcttg
     5641 ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtatt agagtggagg
     5701 tttgacagcc gcctagcatt tcatcacatg gcccgagagc tgcatccgga ctgtactggg
     5761 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     5821 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     5881 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagc
     5941 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6001 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6061 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6121 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6181 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6241 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6301 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6361 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6421 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6481 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6541 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6601 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6661 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6721 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6781 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6841 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6901 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6961 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     7021 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     7081 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7141 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7201 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     7261 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     7321 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     7381 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     7441 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     7501 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7561 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7621 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7681 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7741 gtgccacctg acgtcgacgg
//
