LOCUS       sequence-361931-        7759 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 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-sgStag2#2/Cre
FEATURES             Location/Qualifiers
     source          1..7759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(94..113)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        285..664
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        666..864
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     819..839
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             882..1062
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1109..1234
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1731..1964
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2149..2193
                     /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    2491..2608
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2690..2938
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2690..2710
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2861..2880
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2959..3034
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3087..3586
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3122..3145)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3519..3538
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3590..3610
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3611..4639
                     /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    4695..5283
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4748..4768)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5166..5177)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5286..5302)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5287..5303)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5812..5992
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6054..6642)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6134..6153)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6813..7604,7605..7673))
                     /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     7436..7455
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7674..7759,1..19))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
       61 gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      121 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
      181 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
      241 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      301 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      361 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      421 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      481 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      541 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      601 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      661 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      721 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      781 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      841 acggtgggag gtctatataa gcagcgcgtt ttgcctgtac tgggtctctc tggttagacc
      901 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      961 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     1021 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     1081 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     1141 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     1201 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     1261 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     1321 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     1381 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     1441 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     1501 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     1561 cgcacagcaa gcggccggcc gctgatcttc agacctggag gaggagatat gagggacaat
     1621 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     1681 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     1741 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     1801 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     1861 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     1921 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     1981 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     2041 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     2101 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     2161 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     2221 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     2281 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     2341 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     2401 gagagagaca gagacagatc cattcgatta gtgaacggat cggcactgcg tgcgccaatt
     2461 ctgcagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
     2521 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
     2581 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccagt
     2641 ttggttagta ccgggcccgc tctagccctg caggaactgt ttaaactgtg agggcctatt
     2701 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
     2761 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     2821 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     2881 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac caaacaccgg
     2941 tcaagaagcg ctatgtccgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     3001 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gtttggcgcg cctatggctt
     3061 aattaacatg gcggccgcat tctaccgggt aggggaggcg cttttcccaa ggcagtctgg
     3121 agcatgcgct ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg
     3181 cacacattcc acatccaccg gtaggcgcca accggctccg ttctttggtg gccccttcgc
     3241 gccaccttct actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg
     3301 caggacgtga caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc
     3361 tgagcaatgg aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc
     3421 tttctgggct cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag
     3481 gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg
     3541 cacgtctgcc gcgctgttct cctcttcctc atctccggga cccgggatgc ccaagaagaa
     3601 gaggaaggtg tccaatttac tgaccgtaca ccaaaatttg cctgcattac cggtcgatgc
     3661 aacgagtgat gaggttcgca agaacctgat ggacatgttc agggatcgcc aggcgttttc
     3721 tgagcatacc tggaaaatgc ttctgtccgt ttgccggtcg tgggcggcat ggtgcaagtt
     3781 gaataaccgg aaatggtttc ccgcagaacc tgaagatgtt cgcgattatc ttctatatct
     3841 tcaggcgcgc ggtctggcag taaaaactat ccagcaacat ttgggccagc taaacatgct
     3901 tcatcgtcgg tccgggctgc cacgaccaag tgacagcaat gctgtttcac tggttatgcg
     3961 gcggatccga aaagaaaacg ttgatgccgg tgaacgtgca aaacaggctc tagcgttcga
     4021 acgcactgat ttcgaccagg ttcgttcact catggaaaat agcgatcgct gccaggatat
     4081 acgtaatctg gcatttctgg ggattgctta taacaccctg ttacgtatag ccgaaattgc
     4141 caggatcagg gttaaagata tctcacgtac tgacggtggg agaatgttaa tccatattgg
     4201 cagaacgaaa acgctggtta gcaccgcagg tgtagagaag gcacttagcc tgggggtaac
     4261 taaactggtc gagcgatgga tttccgtctc tggtgtagct gatgatccga ataactacct
     4321 gttttgccgg gtcagaaaaa atggtgttgc cgcgccatct gccaccagcc agctatcaac
     4381 tcgcgccctg gaagggattt ttgaagcaac tcatcgattg atttacggcg ctaaggatga
     4441 ctctggtcag agatacctgg cctggtctgg acacagtgcc cgtgtcggag ccgcgcgaga
     4501 tatggcccgc gctggagttt caataccgga gatcatgcaa gctggtggct ggaccaatgt
     4561 aaatattgtc atgaactata tccgtaacct ggatagtgaa acaggggcaa tggtgcgcct
     4621 gctggaagat ggcgattagc catgtttaag ggttccggtt ccactaggta caattcgata
     4681 tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     4741 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     4801 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     4861 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     4921 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     4981 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     5041 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     5101 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     5161 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     5221 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     5281 cgcatcgata ccgtcgacct cgatcgagac ctagaaaaac atggagcaat cacaagtagc
     5341 aatacagcag ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg
     5401 ggttttccag tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat
     5461 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     5521 caagatatcc ttgatctgtg gatctaccac acacaaggct acttccctga ttggcagaac
     5581 tacacaccag ggccagggat cagatatcca ctgacctttg gatggtgcta caagctagta
     5641 ccagttgagc aagagaaggt agaagaagcc aatgaaggag agaacacccg cttgttacac
     5701 cctgtgagcc tgcatgggat ggatgacccg gagagagaag tattagagtg gaggtttgac
     5761 agccgcctag catttcatca catggcccga gagctgcatc cggactgtac tgggtctctc
     5821 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     5881 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     5941 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagcatgtga
     6001 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     6061 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     6121 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     6181 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     6241 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     6301 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     6361 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     6421 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     6481 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     6541 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     6601 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     6661 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6721 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     6781 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6841 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6901 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6961 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     7021 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     7081 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     7141 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     7201 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     7261 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     7321 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     7381 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     7441 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     7501 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     7561 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     7621 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     7681 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7741 gaatgtattt agaaaaata
//
