LOCUS       sequence-203237-        7437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of Rosa26 targeting sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7437)
  AUTHORS   Roe JS, Hwang CI, Somerville TDD, Milazzo JP, Lee EJ, Da Silva B, 
            Maiorino L, Tiriac H, Young CM, Miyabayashi K, Filippini D, 
            Creighton B, Burkhart RA, Buscaglia JM, Kim EJ, Grem JL, Lazenby AJ,
            Grunkemeyer JA, Hollingsworth MA, Grandgenett PM, Egeblad M, Park Y,
            Tuveson DA, Vakoc CR
  TITLE     Enhancer Reprogramming Promotes Pancreatic Cancer Metastasis.
  JOURNAL   Cell. 2017 Aug 24;170(5):875-888.e20. doi: 
            10.1016/j.cell.2017.07.007. Epub 2017 Jul 27.
  PUBMED    28757253
REFERENCE   2  (bases 1 to 7437)
  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"; doi: 
            "10.1016/j.cell.2017.07.007"; journalName: "Cell"; date: 
            "2017-08-24- 24"; volume: "170"; issue: "5"; pages: "875-888.e20"
COMMENT     Sequence Label: LRG/Rosa26
FEATURES             Location/Qualifiers
     source          1..7437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /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"
     promoter        1713..1961
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1713..1733
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1884..1903
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1982..2057
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    2105..2222
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2291..2502
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2522..2531
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2531..2554
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             join(2555..2557,2558..2560,2561..3274)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2555..2557,2558..2560,2561..3271)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2600..2621)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2861..2880)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3208..3229
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3290..3878
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3343..3363)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3761..3772)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3950..4183
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4261..4382
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4298..4317)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4352..4371
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4422..4557
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4577..4596)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4578..4596)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4606..4623)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4606..4622)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4615..4637)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4764..5219
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4851..4870)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5061..5082
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5245..5349
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5350..5418,5419..6210)
                     /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     complement(5568..5587)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6381..6969
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6870..6889
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7123..7140
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7257..7278
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7293..7298,7299..7316,7317..7323)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7331..7347
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7336..7358
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7355..7371
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7355..7371
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7390..7410
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7392..7410
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggaccca gagagggcct atttcccatg attccttcat
     1741 atttgcatat acgatacaag gctgttagag agataattag aattaatttg actgtaaaca
     1801 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     1861 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     1921 atttcttggc tttatatatc ttgtggaaag gacgaaacac cgaagatggg cgggagtctt
     1981 cgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
     2041 tggcaccgag tcggtgcttt tttgaattct agatcttgag acaaatggca gtattcatcc
     2101 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     2161 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     2221 ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag tggctccggt
     2281 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     2341 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     2401 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     2461 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggatc
     2521 cgccaccatg gattacaaag acgatgacga taagatggtg agcaagggcg aggagctgtt
     2581 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     2641 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     2701 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
     2761 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     2821 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     2881 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     2941 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     3001 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     3061 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     3121 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     3181 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     3241 gatcactctc ggcatggacg agctgtacaa gtagacgcgt taagtcgaca atcaacctct
     3301 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     3361 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     3421 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     3481 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     3541 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     3601 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     3661 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     3721 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     3781 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     3841 tcagacgagt cggatctccc tttgggccgc ctccccgcgt cgactttaag accaatgact
     3901 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     3961 attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac
     4021 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     4081 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     4141 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtacgta tagtagttca
     4201 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
     4261 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4321 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4381 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc
     4441 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4501 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4561 ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
     4621 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     4681 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     4741 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4801 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4861 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4921 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4981 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     5041 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     5101 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     5161 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     5221 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5281 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5341 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5401 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5461 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5521 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5581 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5641 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5701 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5761 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5821 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5881 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5941 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     6001 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6061 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6121 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6181 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6241 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     6301 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     6361 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     6421 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6481 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6541 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6601 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6661 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6721 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6781 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6841 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6901 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6961 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     7021 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     7081 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     7141 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     7201 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     7261 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     7321 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     7381 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaa
//
