LOCUS       sequence-363560-        7760 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses a Smarca4-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 Apr 14, 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-sgSmarca4#2/Cre
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        177..556
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        558..756
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     711..731
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             774..954
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1001..1126
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1623..1856
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2041..2085
                     /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    2383..2500
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2582..2830
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2582..2602
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2753..2772
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2852..2927
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2980..3479
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3015..3038)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3412..3431
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3483..3503
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3504..4532
                     /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    4588..5176
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4641..4661)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5059..5070)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5179..5195)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5180..5196)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5705..5885
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(5947..6535)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6027..6046)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6706..7497,7498..7566))
                     /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     7329..7348
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7567..7671)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(7746..7760,1..5))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
       61 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      121 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      181 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
      241 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
      301 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
      361 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
      421 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
      481 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
      541 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
      601 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
      661 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
      721 cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc
      781 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      841 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      901 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
      961 cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg
     1021 gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat
     1081 tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg
     1141 gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa
     1201 attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct
     1261 gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac
     1321 aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca
     1381 aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa
     1441 aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat
     1501 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1561 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1621 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1681 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1741 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1801 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1861 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1921 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1981 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     2041 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     2101 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     2161 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2221 ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa
     2281 gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg
     2341 cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
     2401 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
     2461 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
     2521 cagagatcca gtttggttag taccgggccc gctctagccc tgcaggaact gtttaaactg
     2581 tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     2641 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     2701 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     2761 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     2821 accaaacacc gcggaggaat cagcttctgt ggttttagag ctagaaatag caagttaaaa
     2881 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgtttggc
     2941 gcgcctatgg cttaattaac atggcggccg cattctaccg ggtaggggag gcgcttttcc
     3001 caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg
     3061 gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg
     3121 gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc
     3181 agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga
     3241 tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc
     3301 tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg ggcgggctca
     3361 ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca
     3421 cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggacccggga
     3481 tgcccaagaa gaagaggaag gtgtccaatt tactgaccgt acaccaaaat ttgcctgcat
     3541 taccggtcga tgcaacgagt gatgaggttc gcaagaacct gatggacatg ttcagggatc
     3601 gccaggcgtt ttctgagcat acctggaaaa tgcttctgtc cgtttgccgg tcgtgggcgg
     3661 catggtgcaa gttgaataac cggaaatggt ttcccgcaga acctgaagat gttcgcgatt
     3721 atcttctata tcttcaggcg cgcggtctgg cagtaaaaac tatccagcaa catttgggcc
     3781 agctaaacat gcttcatcgt cggtccgggc tgccacgacc aagtgacagc aatgctgttt
     3841 cactggttat gcggcggatc cgaaaagaaa acgttgatgc cggtgaacgt gcaaaacagg
     3901 ctctagcgtt cgaacgcact gatttcgacc aggttcgttc actcatggaa aatagcgatc
     3961 gctgccagga tatacgtaat ctggcatttc tggggattgc ttataacacc ctgttacgta
     4021 tagccgaaat tgccaggatc agggttaaag atatctcacg tactgacggt gggagaatgt
     4081 taatccatat tggcagaacg aaaacgctgg ttagcaccgc aggtgtagag aaggcactta
     4141 gcctgggggt aactaaactg gtcgagcgat ggatttccgt ctctggtgta gctgatgatc
     4201 cgaataacta cctgttttgc cgggtcagaa aaaatggtgt tgccgcgcca tctgccacca
     4261 gccagctatc aactcgcgcc ctggaaggga tttttgaagc aactcatcga ttgatttacg
     4321 gcgctaagga tgactctggt cagagatacc tggcctggtc tggacacagt gcccgtgtcg
     4381 gagccgcgcg agatatggcc cgcgctggag tttcaatacc ggagatcatg caagctggtg
     4441 gctggaccaa tgtaaatatt gtcatgaact atatccgtaa cctggatagt gaaacagggg
     4501 caatggtgcg cctgctggaa gatggcgatt agccatgttt aagggttccg gttccactag
     4561 gtacaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga
     4621 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     4681 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     4741 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     4801 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     4861 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     4921 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     4981 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     5041 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     5101 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     5161 tgggccgcct ccccgcatcg ataccgtcga cctcgatcga gacctagaaa aacatggagc
     5221 aatcacaagt agcaatacag cagctaccaa tgctgattgt gcctggctag aagcacaaga
     5281 ggaggaggag gtgggttttc cagtcacacc tcaggtacct ttaagaccaa tgacttacaa
     5341 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     5401 ctcccaacga agacaagata tccttgatct gtggatctac cacacacaag gctacttccc
     5461 tgattggcag aactacacac cagggccagg gatcagatat ccactgacct ttggatggtg
     5521 ctacaagcta gtaccagttg agcaagagaa ggtagaagaa gccaatgaag gagagaacac
     5581 ccgcttgtta caccctgtga gcctgcatgg gatggatgac ccggagagag aagtattaga
     5641 gtggaggttt gacagccgcc tagcatttca tcacatggcc cgagagctgc atccggactg
     5701 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     5761 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     5821 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     5881 tagcagcatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5941 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6001 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6061 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6121 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6181 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6241 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6301 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6361 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     6421 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6481 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6541 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6601 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6661 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6721 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6781 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6841 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6901 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6961 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     7021 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7081 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7141 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7201 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7261 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7321 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     7381 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     7441 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7501 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7561 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     7621 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7681 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
     7741 ctcagtacaa tctgctctga
//
