LOCUS       sequence-361629-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Pole-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-sgPole#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(168..187)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        359..738
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        740..938
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     893..913
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             956..1136
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1183..1308
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1805..2038
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2223..2267
                     /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    2565..2682
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2764..3012
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2764..2784
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2935..2954
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3034..3109
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3162..3661
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3197..3220)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3594..3613
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3665..3685
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3686..4714
                     /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    4770..5358
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4823..4843)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5241..5252)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5361..5377)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5362..5378)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5887..6067
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6129..6717)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6209..6228)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6888..7679,7680..7748))
                     /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     7511..7530
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7749..7760,1..93))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
       61 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
      121 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
      181 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
      241 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      301 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      361 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      421 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      481 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      541 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      601 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      661 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      721 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      781 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      841 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      901 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc
      961 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     1021 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     1081 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     1141 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     1201 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     1261 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     1321 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     1381 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     1441 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     1501 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     1561 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1621 aaaagtaaga ccaccgcaca gcaagcggcc ggccgctgat cttcagacct ggaggaggag
     1681 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     1741 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     1801 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     1861 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     1921 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     1981 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     2041 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     2101 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     2161 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     2221 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     2281 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     2341 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     2401 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     2461 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac
     2521 tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     2581 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     2641 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     2701 agcagagatc cagtttggtt agtaccgggc ccgctctagc cctgcaggaa ctgtttaaac
     2761 tgtgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     2821 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     2881 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     2941 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     3001 ggaccaaaca ccgtcctgag gaacagtgga cgggttttag agctagaaat agcaagttaa
     3061 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttg
     3121 gcgcgcctat ggcttaatta acatggcggc cgcattctac cgggtagggg aggcgctttt
     3181 cccaaggcag tctggagcat gcgctttagc agccccgctg ggcacttggc gctacacaag
     3241 tggcctctgg cctcgcacac attccacatc caccggtagg cgccaaccgg ctccgttctt
     3301 tggtggcccc ttcgcgccac cttctactcc tcccctagtc aggaagttcc cccccgcccc
     3361 gcagctcgcg tcgtgcagga cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca
     3421 gatggacagc accgctgagc aatggaagcg ggtaggcctt tggggcagcg gccaatagca
     3481 gctttgctcc ttcgctttct gggctcagag gctgggaagg ggtgggtccg ggggcgggct
     3541 caggggcggg ctcaggggcg gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg
     3601 cacgcttcaa aagcgcacgt ctgccgcgct gttctcctct tcctcatctc cgggacccgg
     3661 gatgcccaag aagaagagga aggtgtccaa tttactgacc gtacaccaaa atttgcctgc
     3721 attaccggtc gatgcaacga gtgatgaggt tcgcaagaac ctgatggaca tgttcaggga
     3781 tcgccaggcg ttttctgagc atacctggaa aatgcttctg tccgtttgcc ggtcgtgggc
     3841 ggcatggtgc aagttgaata accggaaatg gtttcccgca gaacctgaag atgttcgcga
     3901 ttatcttcta tatcttcagg cgcgcggtct ggcagtaaaa actatccagc aacatttggg
     3961 ccagctaaac atgcttcatc gtcggtccgg gctgccacga ccaagtgaca gcaatgctgt
     4021 ttcactggtt atgcggcgga tccgaaaaga aaacgttgat gccggtgaac gtgcaaaaca
     4081 ggctctagcg ttcgaacgca ctgatttcga ccaggttcgt tcactcatgg aaaatagcga
     4141 tcgctgccag gatatacgta atctggcatt tctggggatt gcttataaca ccctgttacg
     4201 tatagccgaa attgccagga tcagggttaa agatatctca cgtactgacg gtgggagaat
     4261 gttaatccat attggcagaa cgaaaacgct ggttagcacc gcaggtgtag agaaggcact
     4321 tagcctgggg gtaactaaac tggtcgagcg atggatttcc gtctctggtg tagctgatga
     4381 tccgaataac tacctgtttt gccgggtcag aaaaaatggt gttgccgcgc catctgccac
     4441 cagccagcta tcaactcgcg ccctggaagg gatttttgaa gcaactcatc gattgattta
     4501 cggcgctaag gatgactctg gtcagagata cctggcctgg tctggacaca gtgcccgtgt
     4561 cggagccgcg cgagatatgg cccgcgctgg agtttcaata ccggagatca tgcaagctgg
     4621 tggctggacc aatgtaaata ttgtcatgaa ctatatccgt aacctggata gtgaaacagg
     4681 ggcaatggtg cgcctgctgg aagatggcga ttagccatgt ttaagggttc cggttccact
     4741 aggtacaatt cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa
     4801 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     4861 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     4921 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     4981 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     5041 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     5101 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     5161 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     5221 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     5281 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     5341 tttgggccgc ctccccgcat cgataccgtc gacctcgatc gagacctaga aaaacatgga
     5401 gcaatcacaa gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa
     5461 gaggaggagg aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac
     5521 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     5581 cactcccaac gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc
     5641 cctgattggc agaactacac accagggcca gggatcagat atccactgac ctttggatgg
     5701 tgctacaagc tagtaccagt tgagcaagag aaggtagaag aagccaatga aggagagaac
     5761 acccgcttgt tacaccctgt gagcctgcat gggatggatg acccggagag agaagtatta
     5821 gagtggaggt ttgacagccg cctagcattt catcacatgg cccgagagct gcatccggac
     5881 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     5941 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     6001 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     6061 tctagcagca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     6121 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     6181 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6241 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6301 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     6361 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6421 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6481 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6541 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6601 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6661 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6721 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6781 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6841 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6901 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6961 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     7021 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     7081 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     7141 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     7201 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     7261 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     7321 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7381 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7441 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7501 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7561 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7621 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7681 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7741 atactcatac tcttcctttt
//
