LOCUS       sequence-363561-        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#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(133..152)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        324..703
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        705..903
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     858..878
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             921..1101
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1148..1273
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1770..2003
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2188..2232
                     /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    2530..2647
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2729..2977
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2729..2749
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2900..2919
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2999..3074
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3127..3626
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3162..3185)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3559..3578
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3630..3650
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3651..4679
                     /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    4735..5323
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4788..4808)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5206..5217)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5326..5342)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5327..5343)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5852..6032
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6094..6682)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6174..6193)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6853..7644,7645..7713))
                     /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     7476..7495
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7714..7760,1..58))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
       61 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
      121 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
      181 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
      241 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
      301 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
      361 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      421 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      481 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      541 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      601 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      661 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      721 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      781 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      841 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      901 cagcgcgttt tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct
      961 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     1021 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     1081 cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc
     1141 agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg
     1201 gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg
     1261 gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt
     1321 aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct
     1381 agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact
     1441 gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac
     1501 agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt
     1561 agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg
     1621 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     1681 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     1741 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     1801 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     1861 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     1921 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     1981 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     2041 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     2101 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     2161 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     2221 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     2281 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     2341 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     2401 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     2461 attcgattag tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt
     2521 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
     2581 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
     2641 aaattttcgg gtttattaca gggacagcag agatccagtt tggttagtac cgggcccgct
     2701 ctagccctgc aggaactgtt taaactgtga gggcctattt cccatgattc cttcatattt
     2761 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
     2821 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     2881 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     2941 cttggcttta tatatcttgt ggaaaggacc aaacaccgta tgaagtctac acgcaggggt
     3001 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     3061 caccgagtcg gtgctttttt gtttggcgcg cctatggctt aattaacatg gcggccgcat
     3121 tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc
     3181 cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg
     3241 gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttct actcctcccc
     3301 tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag
     3361 tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag
     3421 gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg
     3481 gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa
     3541 ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct
     3601 cctcttcctc atctccggga cccgggatgc ccaagaagaa gaggaaggtg tccaatttac
     3661 tgaccgtaca ccaaaatttg cctgcattac cggtcgatgc aacgagtgat gaggttcgca
     3721 agaacctgat ggacatgttc agggatcgcc aggcgttttc tgagcatacc tggaaaatgc
     3781 ttctgtccgt ttgccggtcg tgggcggcat ggtgcaagtt gaataaccgg aaatggtttc
     3841 ccgcagaacc tgaagatgtt cgcgattatc ttctatatct tcaggcgcgc ggtctggcag
     3901 taaaaactat ccagcaacat ttgggccagc taaacatgct tcatcgtcgg tccgggctgc
     3961 cacgaccaag tgacagcaat gctgtttcac tggttatgcg gcggatccga aaagaaaacg
     4021 ttgatgccgg tgaacgtgca aaacaggctc tagcgttcga acgcactgat ttcgaccagg
     4081 ttcgttcact catggaaaat agcgatcgct gccaggatat acgtaatctg gcatttctgg
     4141 ggattgctta taacaccctg ttacgtatag ccgaaattgc caggatcagg gttaaagata
     4201 tctcacgtac tgacggtggg agaatgttaa tccatattgg cagaacgaaa acgctggtta
     4261 gcaccgcagg tgtagagaag gcacttagcc tgggggtaac taaactggtc gagcgatgga
     4321 tttccgtctc tggtgtagct gatgatccga ataactacct gttttgccgg gtcagaaaaa
     4381 atggtgttgc cgcgccatct gccaccagcc agctatcaac tcgcgccctg gaagggattt
     4441 ttgaagcaac tcatcgattg atttacggcg ctaaggatga ctctggtcag agatacctgg
     4501 cctggtctgg acacagtgcc cgtgtcggag ccgcgcgaga tatggcccgc gctggagttt
     4561 caataccgga gatcatgcaa gctggtggct ggaccaatgt aaatattgtc atgaactata
     4621 tccgtaacct ggatagtgaa acaggggcaa tggtgcgcct gctggaagat ggcgattagc
     4681 catgtttaag ggttccggtt ccactaggta caattcgata tcaagcttat cgataatcaa
     4741 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     4801 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     4861 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     4921 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     4981 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     5041 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     5101 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     5161 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     5221 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     5281 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgacct
     5341 cgatcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc
     5401 tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca
     5461 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     5521 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg
     5581 gatctaccac acacaaggct acttccctga ttggcagaac tacacaccag ggccagggat
     5641 cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc aagagaaggt
     5701 agaagaagcc aatgaaggag agaacacccg cttgttacac cctgtgagcc tgcatgggat
     5761 ggatgacccg gagagagaag tattagagtg gaggtttgac agccgcctag catttcatca
     5821 catggcccga gagctgcatc cggactgtac tgggtctctc tggttagacc agatctgagc
     5881 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     5941 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     6001 acccttttag tcagtgtgga aaatctctag cagcatgtga gcaaaaggcc agcaaaaggc
     6061 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6121 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6181 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6241 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     6301 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6361 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6421 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     6481 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     6541 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     6601 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     6661 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6721 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6781 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6841 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6901 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6961 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     7021 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7081 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7141 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7201 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7261 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7321 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7381 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7441 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     7501 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     7561 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     7621 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     7681 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7741 catttatcag ggttattgtc
//
