LOCUS       sequence-361932-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a Stag2-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-sgStag2#1/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(159..178)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        350..729
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        731..929
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     884..904
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             947..1127
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1174..1299
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1796..2029
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2214..2258
                     /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    2556..2673
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2755..3003
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2755..2775
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2926..2945
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3025..3100
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3153..3652
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3188..3211)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3585..3604
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3656..3676
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3677..4705
                     /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    4761..5349
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4814..4834)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5232..5243)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5352..5368)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5353..5369)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5878..6058
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6120..6708)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6200..6219)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6879..7670,7671..7739))
                     /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     7502..7521
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7740..7760,1..84))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
       61 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
      121 atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca
      181 tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc
      241 aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg
      301 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
      361 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      421 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      481 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      541 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      601 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      661 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      721 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      781 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      841 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      901 cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt
      961 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     1021 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     1081 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac
     1141 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct
     1201 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact
     1261 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt
     1321 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa
     1381 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa
     1441 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca
     1501 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata
     1561 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag
     1621 accaccgcac agcaagcggc cggccgctga tcttcagacc tggaggagga gatatgaggg
     1681 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1741 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1801 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     1861 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1921 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1981 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     2041 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     2101 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     2161 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     2221 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     2281 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     2341 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     2401 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
     2461 gtggagagag agacagagac agatccattc gattagtgaa cggatcggca ctgcgtgcgc
     2521 caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg
     2581 gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa
     2641 ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat
     2701 ccagtttggt tagtaccggg cccgctctag ccctgcagga actgtttaaa ctgtgagggc
     2761 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     2821 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     2881 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     2941 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggaccaaac
     3001 accgcgagtc ctacaagcat gaccgtttta gagctagaaa tagcaagtta aaataaggct
     3061 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttgttt ggcgcgccta
     3121 tggcttaatt aacatggcgg ccgcattcta ccgggtaggg gaggcgcttt tcccaaggca
     3181 gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg
     3241 gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc
     3301 cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc
     3361 gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag
     3421 caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc
     3481 cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg
     3541 gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca
     3601 aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggacccg ggatgcccaa
     3661 gaagaagagg aaggtgtcca atttactgac cgtacaccaa aatttgcctg cattaccggt
     3721 cgatgcaacg agtgatgagg ttcgcaagaa cctgatggac atgttcaggg atcgccaggc
     3781 gttttctgag catacctgga aaatgcttct gtccgtttgc cggtcgtggg cggcatggtg
     3841 caagttgaat aaccggaaat ggtttcccgc agaacctgaa gatgttcgcg attatcttct
     3901 atatcttcag gcgcgcggtc tggcagtaaa aactatccag caacatttgg gccagctaaa
     3961 catgcttcat cgtcggtccg ggctgccacg accaagtgac agcaatgctg tttcactggt
     4021 tatgcggcgg atccgaaaag aaaacgttga tgccggtgaa cgtgcaaaac aggctctagc
     4081 gttcgaacgc actgatttcg accaggttcg ttcactcatg gaaaatagcg atcgctgcca
     4141 ggatatacgt aatctggcat ttctggggat tgcttataac accctgttac gtatagccga
     4201 aattgccagg atcagggtta aagatatctc acgtactgac ggtgggagaa tgttaatcca
     4261 tattggcaga acgaaaacgc tggttagcac cgcaggtgta gagaaggcac ttagcctggg
     4321 ggtaactaaa ctggtcgagc gatggatttc cgtctctggt gtagctgatg atccgaataa
     4381 ctacctgttt tgccgggtca gaaaaaatgg tgttgccgcg ccatctgcca ccagccagct
     4441 atcaactcgc gccctggaag ggatttttga agcaactcat cgattgattt acggcgctaa
     4501 ggatgactct ggtcagagat acctggcctg gtctggacac agtgcccgtg tcggagccgc
     4561 gcgagatatg gcccgcgctg gagtttcaat accggagatc atgcaagctg gtggctggac
     4621 caatgtaaat attgtcatga actatatccg taacctggat agtgaaacag gggcaatggt
     4681 gcgcctgctg gaagatggcg attagccatg tttaagggtt ccggttccac taggtacaat
     4741 tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     4801 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     4861 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     4921 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     4981 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     5041 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     5101 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     5161 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     5221 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     5281 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     5341 cctccccgca tcgataccgt cgacctcgat cgagacctag aaaaacatgg agcaatcaca
     5401 agtagcaata cagcagctac caatgctgat tgtgcctggc tagaagcaca agaggaggag
     5461 gaggtgggtt ttccagtcac acctcaggta cctttaagac caatgactta caaggcagct
     5521 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     5581 cgaagacaag atatccttga tctgtggatc taccacacac aaggctactt ccctgattgg
     5641 cagaactaca caccagggcc agggatcaga tatccactga cctttggatg gtgctacaag
     5701 ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg aaggagagaa cacccgcttg
     5761 ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtatt agagtggagg
     5821 tttgacagcc gcctagcatt tcatcacatg gcccgagagc tgcatccgga ctgtactggg
     5881 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     5941 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     6001 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagc
     6061 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6121 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6181 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6241 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6301 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6361 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6421 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6481 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6541 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6601 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6661 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6721 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6781 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6841 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6901 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6961 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     7021 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     7081 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     7141 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     7201 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7261 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7321 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     7381 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     7441 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     7501 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     7561 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     7621 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7681 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7741 ctcttccttt ttcaatatta
//
