LOCUS       sequence-362055-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Brca2-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7760)
  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 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: "Cancer 
            Discov. 2021 Feb 19. pii: 2159-8290.CD-20-1325. doi: 
            10.1158/2159-8290.CD-20-1325."
COMMENT     Sequence Label: Lenti-sgBrca2#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(122..141)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        313..692
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        694..892
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     847..867
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             910..1090
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1137..1262
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1759..1992
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2177..2221
                     /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    2519..2636
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2718..2966
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2718..2738
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2889..2908
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2988..3063
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3116..3615
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3151..3174)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3548..3567
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3619..3639
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3640..4668
                     /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    4724..5312
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4777..4797)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5195..5206)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5315..5331)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5316..5332)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5841..6021
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6083..6671)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6163..6182)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6842..7633,7634..7702))
                     /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     7465..7484
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7703..7760,1..47))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
       61 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
      121 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
      181 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
      241 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
      301 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      361 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      421 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      481 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      541 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      601 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      661 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      721 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      781 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      841 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agcgcgtttt
      901 gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
      961 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     1021 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     1081 atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct
     1141 ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg
     1201 tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg
     1261 tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg
     1321 gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg
     1381 cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac
     1441 aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc
     1501 tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag
     1561 aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccggccgc tgatcttcag
     1621 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
     1681 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
     1741 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
     1801 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
     1861 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
     1921 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
     1981 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
     2041 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
     2101 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
     2161 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
     2221 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
     2281 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
     2341 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag
     2401 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt
     2461 gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc atccacaatt
     2521 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     2581 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     2641 tttattacag ggacagcaga gatccagttt ggttagtacc gggcccgctc tagccctgca
     2701 ggaactgttt aaactgtgag ggcctatttc ccatgattcc ttcatatttg catatacgat
     2761 acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag atattagtac
     2821 aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta aaattatgtt
     2881 ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc ttggctttat
     2941 atatcttgtg gaaaggacca aacaccgctg aaaggcgact ggtccaggtt ttagagctag
     3001 aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
     3061 tgcttttttg tttggcgcgc ctatggctta attaacatgg cggccgcatt ctaccgggta
     3121 ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact
     3181 tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa
     3241 ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag
     3301 ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc
     3361 actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc
     3421 agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg
     3481 tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga
     3541 ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca
     3601 tctccgggac ccgggatgcc caagaagaag aggaaggtgt ccaatttact gaccgtacac
     3661 caaaatttgc ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg
     3721 gacatgttca gggatcgcca ggcgttttct gagcatacct ggaaaatgct tctgtccgtt
     3781 tgccggtcgt gggcggcatg gtgcaagttg aataaccgga aatggtttcc cgcagaacct
     3841 gaagatgttc gcgattatct tctatatctt caggcgcgcg gtctggcagt aaaaactatc
     3901 cagcaacatt tgggccagct aaacatgctt catcgtcggt ccgggctgcc acgaccaagt
     3961 gacagcaatg ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt
     4021 gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc
     4081 atggaaaata gcgatcgctg ccaggatata cgtaatctgg catttctggg gattgcttat
     4141 aacaccctgt tacgtatagc cgaaattgcc aggatcaggg ttaaagatat ctcacgtact
     4201 gacggtggga gaatgttaat ccatattggc agaacgaaaa cgctggttag caccgcaggt
     4261 gtagagaagg cacttagcct gggggtaact aaactggtcg agcgatggat ttccgtctct
     4321 ggtgtagctg atgatccgaa taactacctg ttttgccggg tcagaaaaaa tggtgttgcc
     4381 gcgccatctg ccaccagcca gctatcaact cgcgccctgg aagggatttt tgaagcaact
     4441 catcgattga tttacggcgc taaggatgac tctggtcaga gatacctggc ctggtctgga
     4501 cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc aataccggag
     4561 atcatgcaag ctggtggctg gaccaatgta aatattgtca tgaactatat ccgtaacctg
     4621 gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg gcgattagcc atgtttaagg
     4681 gttccggttc cactaggtac aattcgatat caagcttatc gataatcaac ctctggatta
     4741 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     4801 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     4861 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     4921 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     4981 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     5041 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     5101 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     5161 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     5221 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     5281 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc
     5341 tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct
     5401 ggctagaagc acaagaggag gaggaggtgg gttttccagt cacacctcag gtacctttaa
     5461 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     5521 tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca
     5581 cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac
     5641 tgacctttgg atggtgctac aagctagtac cagttgagca agagaaggta gaagaagcca
     5701 atgaaggaga gaacacccgc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
     5761 agagagaagt attagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag
     5821 agctgcatcc ggactgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     5881 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     5941 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     6001 cagtgtggaa aatctctagc agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     6061 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     6121 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     6181 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     6241 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     6301 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6361 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6421 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6481 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     6541 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6601 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6661 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6721 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6781 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6841 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6901 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6961 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     7021 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     7081 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     7141 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     7201 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     7261 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     7321 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     7381 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     7441 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     7501 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7561 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7621 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7681 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7741 gttattgtct catgagcgga
//
