LOCUS       sequence-361626-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Ercc4-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-sgErcc4#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(161..180)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        352..731
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        733..931
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     886..906
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             949..1129
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1176..1301
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1798..2031
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2216..2260
                     /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    2558..2675
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2757..3005
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2757..2777
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2928..2947
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3027..3102
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3155..3654
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3190..3213)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3587..3606
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3658..3678
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3679..4707
                     /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    4763..5351
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4816..4836)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5234..5245)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5354..5370)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5355..5371)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5880..6060
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6122..6710)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6202..6221)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6881..7672,7673..7741))
                     /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     7504..7523
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7742..7760,1..86))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
       61 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
      121 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
      181 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
      241 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
      301 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat
      361 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      421 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      481 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      541 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      601 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      661 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      721 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      781 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      841 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      901 ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg
      961 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     1021 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     1081 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
     1141 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
     1201 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
     1261 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
     1321 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
     1381 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
     1441 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
     1501 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1561 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1621 agaccaccgc acagcaagcg gccggccgct gatcttcaga cctggaggag gagatatgag
     1681 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1741 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1801 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cgtcaatgac
     1861 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1921 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1981 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     2041 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     2101 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     2161 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     2221 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     2281 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     2341 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     2401 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     2461 aggtggagag agagacagag acagatccat tcgattagtg aacggatcgg cactgcgtgc
     2521 gccaattctg cagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg
     2581 gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag
     2641 aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag
     2701 atccagtttg gttagtaccg ggcccgctct agccctgcag gaactgttta aactgtgagg
     2761 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     2821 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     2881 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     2941 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggaccaa
     3001 acaccgacaa agggtccaag taatgggttt tagagctaga aatagcaagt taaaataagg
     3061 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgt ttggcgcgcc
     3121 tatggcttaa ttaacatggc ggccgcattc taccgggtag gggaggcgct tttcccaagg
     3181 cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc
     3241 tggcctcgca cacattccac atccaccggt aggcgccaac cggctccgtt ctttggtggc
     3301 cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc
     3361 gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac
     3421 agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc
     3481 tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc
     3541 gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt
     3601 caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggacc cgggatgccc
     3661 aagaagaaga ggaaggtgtc caatttactg accgtacacc aaaatttgcc tgcattaccg
     3721 gtcgatgcaa cgagtgatga ggttcgcaag aacctgatgg acatgttcag ggatcgccag
     3781 gcgttttctg agcatacctg gaaaatgctt ctgtccgttt gccggtcgtg ggcggcatgg
     3841 tgcaagttga ataaccggaa atggtttccc gcagaacctg aagatgttcg cgattatctt
     3901 ctatatcttc aggcgcgcgg tctggcagta aaaactatcc agcaacattt gggccagcta
     3961 aacatgcttc atcgtcggtc cgggctgcca cgaccaagtg acagcaatgc tgtttcactg
     4021 gttatgcggc ggatccgaaa agaaaacgtt gatgccggtg aacgtgcaaa acaggctcta
     4081 gcgttcgaac gcactgattt cgaccaggtt cgttcactca tggaaaatag cgatcgctgc
     4141 caggatatac gtaatctggc atttctgggg attgcttata acaccctgtt acgtatagcc
     4201 gaaattgcca ggatcagggt taaagatatc tcacgtactg acggtgggag aatgttaatc
     4261 catattggca gaacgaaaac gctggttagc accgcaggtg tagagaaggc acttagcctg
     4321 ggggtaacta aactggtcga gcgatggatt tccgtctctg gtgtagctga tgatccgaat
     4381 aactacctgt tttgccgggt cagaaaaaat ggtgttgccg cgccatctgc caccagccag
     4441 ctatcaactc gcgccctgga agggattttt gaagcaactc atcgattgat ttacggcgct
     4501 aaggatgact ctggtcagag atacctggcc tggtctggac acagtgcccg tgtcggagcc
     4561 gcgcgagata tggcccgcgc tggagtttca ataccggaga tcatgcaagc tggtggctgg
     4621 accaatgtaa atattgtcat gaactatatc cgtaacctgg atagtgaaac aggggcaatg
     4681 gtgcgcctgc tggaagatgg cgattagcca tgtttaaggg ttccggttcc actaggtaca
     4741 attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgac
     4801 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
     4861 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
     4921 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
     4981 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
     5041 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
     5101 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
     5161 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
     5221 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
     5281 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
     5341 cgcctccccg catcgatacc gtcgacctcg atcgagacct agaaaaacat ggagcaatca
     5401 caagtagcaa tacagcagct accaatgctg attgtgcctg gctagaagca caagaggagg
     5461 aggaggtggg ttttccagtc acacctcagg tacctttaag accaatgact tacaaggcag
     5521 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     5581 aacgaagaca agatatcctt gatctgtgga tctaccacac acaaggctac ttccctgatt
     5641 ggcagaacta cacaccaggg ccagggatca gatatccact gacctttgga tggtgctaca
     5701 agctagtacc agttgagcaa gagaaggtag aagaagccaa tgaaggagag aacacccgct
     5761 tgttacaccc tgtgagcctg catgggatgg atgacccgga gagagaagta ttagagtgga
     5821 ggtttgacag ccgcctagca tttcatcaca tggcccgaga gctgcatccg gactgtactg
     5881 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     5941 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     6001 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     6061 gcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     6121 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6181 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6241 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6301 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6361 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6421 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6481 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6541 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     6601 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6661 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     6721 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6781 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6841 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6901 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6961 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     7021 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     7081 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     7141 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     7201 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7261 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7321 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7381 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7441 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7501 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7561 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7621 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7681 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     7741 tactcttcct ttttcaatat
//
