LOCUS       sequence-363583-        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#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     93..113
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             156..336
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    383..508
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1005..1238
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1423..1467
                     /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    1765..1882
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1964..2212
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1964..1984
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2135..2154
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2234..2309
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2362..2861
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2397..2420)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2794..2813
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2865..2885
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2886..3914
                     /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    3970..4558
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4023..4043)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4441..4452)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4561..4577)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4562..4578)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5087..5267
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5329..5917)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5409..5428)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6088..6879,6880..6948))
                     /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     6711..6730
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6949..7053)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7128..7147)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7319..7698
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(7700..7760,1..138)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
       61 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      121 ggaggtctat ataagcagcg cgttttgcct gtactgggtc tctctggtta gaccagatct
      181 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
      241 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
      301 tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa
      361 agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg aagcgcgcac
      421 ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta gcggaggcta
      481 gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta gatcgcgatg
      541 ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac atatagtatg
      601 ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa catcagaagg
      661 ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag aagaacttag
      721 atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag agataaaaga
      781 caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga ccaccgcaca
      841 gcaagcggcc ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga
      901 agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag
      961 gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt
     1021 gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag
     1081 gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag
     1141 gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc
     1201 ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga
     1261 aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa
     1321 cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc
     1381 ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta
     1441 ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg
     1501 tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct
     1561 gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac
     1621 ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga
     1681 gacagagaca gatccattcg attagtgaac ggatcggcac tgcgtgcgcc aattctgcag
     1741 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
     1801 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
     1861 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cagtttggtt
     1921 agtaccgggc ccgctctagc cctgcaggaa ctgtttaaac tgtgagggcc tatttcccat
     1981 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
     2041 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     2101 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     2161 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggaccaaaca ccgcattatg
     2221 gtcacctaca atggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
     2281 aacttgaaaa agtggcaccg agtcggtgct tttttgtttg gcgcgcctat ggcttaatta
     2341 acatggcggc cgcattctac cgggtagggg aggcgctttt cccaaggcag tctggagcat
     2401 gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac
     2461 attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac
     2521 cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga
     2581 cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc
     2641 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct
     2701 gggctcagag gctgggaagg ggtgggtccg ggggcnngct caggggcggg ctcaggggcg
     2761 gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt
     2821 ctgccgcgct gttctcctct tcctcatctc cgggacccgg gatgcccaag aagaagagga
     2881 aggtgtccaa tttactgacc gtacaccaaa atttgcctgc attaccggtc gatgcaacga
     2941 gtgatgaggt tcgcaagaac ctgatggaca tgttcaggga tcgccaggcg ttttctgagc
     3001 atacctggaa aatgcttctg tccgtttgcc ggtcgtgggc ggcatggtgc aagttgaata
     3061 accggaaatg gtttcccgca gaacctgaag atgttcgcga ttatcttcta tatcttcagg
     3121 cgcgcggtct ggcagtaaaa actatccagc aacatttggg ccagctaaac atgcttcatc
     3181 gtcggtccgg gctgccacga ccaagtgaca gcaatgctgt ttcactggtt atgcggcgga
     3241 tccgaaaaga aaacgttgat gccggtgaac gtgcaaaaca ggctctagcg ttcgaacgca
     3301 ctgatttcga ccaggttcgt tcactcatgg aaaatagcga tcgctgccag gatatacgta
     3361 atctggcatt tctggggatt gcttataaca ccctgttacg tatagccgaa attgccagga
     3421 tcagggttaa agatatctca cgtactgacg gtgggagaat gttaatccat attggcagaa
     3481 cgaaaacgct ggttagcacc gcaggtgtag agaaggcact tagcctgggg gtaactaaac
     3541 tggtcgagcg atggatttcc gtctctggtg tagctgatga tccgaataac tacctgtttt
     3601 gccgggtcag aaaaaatggt gttgccgcgc catctgccac cagccagcta tcaactcgcg
     3661 ccctggaagg gatttttgaa gcaactcatc gattgattta cggcgctaag gatgactctg
     3721 gtcagagata cctggcctgg tctggacaca gtgcccgtgt cggagccgcg cgagatatgg
     3781 cccgcgctgg agtttcaata ccggagatca tgcaagctgg tggctggacc aatgtaaata
     3841 ttgtcatgaa ctatatccgt aacctggata gtgaaacagg ggcaatggtg cgcctgctgg
     3901 aagatggcga ttagccatgt ttaagggttc cggttccact aggtacaatt cgatatcaag
     3961 cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     4021 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     4081 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     4141 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     4201 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     4261 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     4321 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     4381 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     4441 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     4501 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat
     4561 cgataccgtc gacctcgatc gagacctaga aaaacatgga gcaatcacaa gtagcaatac
     4621 agcagctacc aatgctgatt gtgcctggct agaagcacaa gaggaggagg aggtgggttt
     4681 tccagtcaca cctcaggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
     4741 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     4801 tatccttgat ctgtggatct accacacaca aggctacttc cctgattggc agaactacac
     4861 accagggcca gggatcagat atccactgac ctttggatgg tgctacaagc tagtaccagt
     4921 tgagcaagag aaggtagaag aagccaatga aggagagaac acccgcttgt tacaccctgt
     4981 gagcctgcat gggatggatg acccggagag agaagtatta gagtggaggt ttgacagccg
     5041 cctagcattt catcacatgg cccgagagct gcatccggac tgtactgggt ctctctggtt
     5101 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     5161 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     5221 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagca tgtgagcaaa
     5281 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5341 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5401 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5461 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5521 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5581 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5641 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5701 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5761 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5821 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5881 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5941 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6001 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6061 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6121 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6181 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6241 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6301 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6361 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6421 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6481 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6541 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6601 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6661 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6721 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6781 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6841 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6901 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6961 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7021 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     7081 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     7141 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     7201 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     7261 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
     7321 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     7381 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     7441 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     7501 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     7561 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     7621 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     7681 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     7741 tttgactcac ggggatttcc
//
