LOCUS       sequence-176169-        7761 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Neomycin-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7761)
  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 7761)
  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-sgNeo3/Cre
FEATURES             Location/Qualifiers
     source          1..7761
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1686..1919
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2104..2148
                     /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    2446..2563
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2645..2893
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2645..2665
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2816..2835
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2914..2989
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3042..3541
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3077..3100)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3474..3493
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3545..3565
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3566..4594
                     /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    4650..5238
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4703..4723)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5121..5132)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5241..5257)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5242..5258)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5767..5947
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6009..6597)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6089..6108)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6768..7559,7560..7628))
                     /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     7391..7410
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7629..7733)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgcgctga tcttcagacc tggaggagga
     1561 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     1621 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     1681 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     1741 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     1801 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1861 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1921 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1981 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     2041 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     2101 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     2161 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     2221 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     2281 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     2341 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     2401 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     2461 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2521 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2581 cagcagagat ccagtttggt tagtaccggg cccgctctag ccctgcagga actgtttaaa
     2641 ctgtgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     2701 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     2761 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     2821 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     2881 aggaccaaac accgaatagc ctctccaccc aaggttttag agctagaaat agcaagttaa
     2941 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttg
     3001 gcgcgcctat ggcttaatta acatggcggc cgcattctac cgggtagggg aggcgctttt
     3061 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag
     3121 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     3181 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc
     3241 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     3301 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     3361 gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct
     3421 caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg
     3481 cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggacccgg
     3541 gatgcccaag aagaagagga aggtgtccaa tttactgacc gtacaccaaa atttgcctgc
     3601 attaccggtc gatgcaacga gtgatgaggt tcgcaagaac ctgatggaca tgttcaggga
     3661 tcgccaggcg ttttctgagc atacctggaa aatgcttctg tccgtttgcc ggtcgtgggc
     3721 ggcatggtgc aagttgaata accggaaatg gtttcccgca gaacctgaag atgttcgcga
     3781 ttatcttcta tatcttcagg cgcgcggtct ggcagtaaaa actatccagc aacatttggg
     3841 ccagctaaac atgcttcatc gtcggtccgg gctgccacga ccaagtgaca gcaatgctgt
     3901 ttcactggtt atgcggcgga tccgaaaaga aaacgttgat gccggtgaac gtgcaaaaca
     3961 ggctctagcg ttcgaacgca ctgatttcga ccaggttcgt tcactcatgg aaaatagcga
     4021 tcgctgccag gatatacgta atctggcatt tctggggatt gcttataaca ccctgttacg
     4081 tatagccgaa attgccagga tcagggttaa agatatctca cgtactgacg gtgggagaat
     4141 gttaatccat attggcagaa cgaaaacgct ggttagcacc gcaggtgtag agaaggcact
     4201 tagcctgggg gtaactaaac tggtcgagcg atggatttcc gtctctggtg tagctgatga
     4261 tccgaataac tacctgtttt gccgggtcag aaaaaatggt gttgccgcgc catctgccac
     4321 cagccagcta tcaactcgcg ccctggaagg gatttttgaa gcaactcatc gattgattta
     4381 cggcgctaag gatgactctg gtcagagata cctggcctgg tctggacaca gtgcccgtgt
     4441 cggagccgcg cgagatatgg cccgcgctgg agtttcaata ccggagatca tgcaagctgg
     4501 tggctggacc aatgtaaata ttgtcatgaa ctatatccgt aacctggata gtgaaacagg
     4561 ggcaatggtg cgcctgctgg aagatggcga ttagccatgt ttaagggttc cggttccact
     4621 aggtacaatt cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa
     4681 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     4741 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     4801 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     4861 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     4921 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     4981 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     5041 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     5101 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     5161 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     5221 tttgggccgc ctccccgcat cgataccgtc gacctcgatc gagacctaga aaaacatgga
     5281 gcaatcacaa gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa
     5341 gaggaggagg aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac
     5401 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     5461 cactcccaac gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc
     5521 cctgattggc agaactacac accagggcca gggatcagat atccactgac ctttggatgg
     5581 tgctacaagc tagtaccagt tgagcaagag aaggtagaag aagccaatga aggagagaac
     5641 acccgcttgt tacaccctgt gagcctgcat gggatggatg acccggagag agaagtatta
     5701 gagtggaggt ttgacagccg cctagcattt catcacatgg cccgagagct gcatccggac
     5761 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     5821 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     5881 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     5941 tctagcagca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     6001 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     6061 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6121 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6181 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     6241 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6301 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6361 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6421 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6481 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6541 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6601 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6661 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6721 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6781 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6841 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6901 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6961 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     7021 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     7081 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     7141 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     7201 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7261 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7321 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7381 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7441 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7501 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7561 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7621 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7681 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7741 ccccgaaaag tgccacctga c
//
