LOCUS       sequence-362052-        7759 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Fat1-targeting gRNA and Cre-recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7759)
  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 7759)
  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-sgFat1#2/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(132..151)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        323..702
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        704..902
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     857..877
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             920..1100
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1147..1272
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1769..2002
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2187..2231
                     /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    2529..2646
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2728..2976
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2728..2748
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2899..2918
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2997..3072
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3125..3624
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3160..3183)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3557..3576
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3628..3648
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3649..4677
                     /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    4733..5321
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4786..4806)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5204..5215)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5324..5340)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5325..5341)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5850..6030
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6092..6680)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6172..6191)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6851..7642,7643..7711))
                     /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     7474..7493
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7712..7759,1..57))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
       61 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
      121 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
      181 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
      241 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
      301 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
      361 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      421 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      481 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      541 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      601 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      661 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      721 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      781 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      841 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      901 agcgcgtttt gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
      961 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     1021 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     1081 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
     1141 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
     1201 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
     1261 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
     1321 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
     1381 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
     1441 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
     1501 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
     1561 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccggccgc
     1621 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
     1681 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
     1741 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
     1801 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
     1861 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
     1921 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
     1981 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
     2041 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
     2101 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
     2161 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
     2221 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
     2281 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga
     2341 atagagttag gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg
     2401 gacccgacag gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca
     2461 ttcgattagt gaacggatcg gcactgcgtg cgccaattct gcagacaaat ggcagtattc
     2521 atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     2581 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     2641 aattttcggg tttattacag ggacagcaga gatccagttt ggttagtacc gggcccgctc
     2701 tagccctgca ggaactgttt aaactgtgag ggcctatttc ccatgattcc ttcatatttg
     2761 catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag
     2821 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
     2881 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
     2941 ttggctttat atatcttgtg gaaaggacca aacaccgtct catcacaacc acgtcggttt
     3001 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     3061 ccgagtcggt gcttttttgt ttggcgcgcc tatggcttaa ttaacatggc ggccgcattc
     3121 taccgggtag gggaggcgct tttcccaagg cagtctggag catgcgcttt agcagccccg
     3181 ctgggcactt ggcgctacac aagtggcctc tggcctcgca cacattccac atccaccggt
     3241 aggcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta
     3301 gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta
     3361 gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc
     3421 ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga
     3481 aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcccgaagg
     3541 tcctccggag gcccggcatt ctgcacgctt caaaagcgca cgtctgccgc gctgttctcc
     3601 tcttcctcat ctccgggacc cgggatgccc aagaagaaga ggaaggtgtc caatttactg
     3661 accgtacacc aaaatttgcc tgcattaccg gtcgatgcaa cgagtgatga ggttcgcaag
     3721 aacctgatgg acatgttcag ggatcgccag gcgttttctg agcatacctg gaaaatgctt
     3781 ctgtccgttt gccggtcgtg ggcggcatgg tgcaagttga ataaccggaa atggtttccc
     3841 gcagaacctg aagatgttcg cgattatctt ctatatcttc aggcgcgcgg tctggcagta
     3901 aaaactatcc agcaacattt gggccagcta aacatgcttc atcgtcggtc cgggctgcca
     3961 cgaccaagtg acagcaatgc tgtttcactg gttatgcggc ggatccgaaa agaaaacgtt
     4021 gatgccggtg aacgtgcaaa acaggctcta gcgttcgaac gcactgattt cgaccaggtt
     4081 cgttcactca tggaaaatag cgatcgctgc caggatatac gtaatctggc atttctgggg
     4141 attgcttata acaccctgtt acgtatagcc gaaattgcca ggatcagggt taaagatatc
     4201 tcacgtactg acggtgggag aatgttaatc catattggca gaacgaaaac gctggttagc
     4261 accgcaggtg tagagaaggc acttagcctg ggggtaacta aactggtcga gcgatggatt
     4321 tccgtctctg gtgtagctga tgatccgaat aactacctgt tttgccgggt cagaaaaaat
     4381 ggtgttgccg cgccatctgc caccagccag ctatcaactc gcgccctgga agggattttt
     4441 gaagcaactc atcgattgat ttacggcgct aaggatgact ctggtcagag atacctggcc
     4501 tggtctggac acagtgcccg tgtcggagcc gcgcgagata tggcccgcgc tggagtttca
     4561 ataccggaga tcatgcaagc tggtggctgg accaatgtaa atattgtcat gaactatatc
     4621 cgtaacctgg atagtgaaac aggggcaatg gtgcgcctgc tggaagatgg cgattagcca
     4681 tgtttaaggg ttccggttcc actaggtaca attcgatatc aagcttatcg ataatcaacc
     4741 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     4801 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     4861 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     4921 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     4981 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     5041 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     5101 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     5161 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     5221 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     5281 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacctcg
     5341 atcgagacct agaaaaacat ggagcaatca caagtagcaa tacagcagct accaatgctg
     5401 attgtgcctg gctagaagca caagaggagg aggaggtggg ttttccagtc acacctcagg
     5461 tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     5521 aggggggact ggaagggcta attcactccc aacgaagaca agatatcctt gatctgtgga
     5581 tctaccacac acaaggctac ttccctgatt ggcagaacta cacaccaggg ccagggatca
     5641 gatatccact gacctttgga tggtgctaca agctagtacc agttgagcaa gagaaggtag
     5701 aagaagccaa tgaaggagag aacacccgct tgttacaccc tgtgagcctg catgggatgg
     5761 atgacccgga gagagaagta ttagagtgga ggtttgacag ccgcctagca tttcatcaca
     5821 tggcccgaga gctgcatccg gactgtactg ggtctctctg gttagaccag atctgagcct
     5881 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     5941 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     6001 ccttttagtc agtgtggaaa atctctagca gcatgtgagc aaaaggccag caaaaggcca
     6061 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6121 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6181 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6241 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6301 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6361 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6421 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6481 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6541 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6601 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6661 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6721 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6781 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6841 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6901 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6961 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7021 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7081 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7141 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     7201 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7261 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7321 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7381 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7441 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7501 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7561 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7621 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7681 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7741 tttatcaggg ttattgtct
//
