LOCUS       sequence-274413-        8101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lenti expression of mCherry2 with U6 promoter for SauCas9 sgRNA 
            targeting GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8101)
  AUTHORS   Watters KE, Shivram H, Fellmann C, Lew RJ, McMahon B, Doudna JA
  TITLE     Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 Mar 10. pii: 1917668117. doi: 
            10.1073/pnas.1917668117.
  PUBMED    32156733
REFERENCE   2  (bases 1 to 8101)
  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: "Proc Natl 
            Acad Sci U S A. 2020 Mar 10. pii: 1917668117. doi: 
            10.1073/pnas.1917668117."
COMMENT     Sequence Label: pCF824_U6-Sau-sgRNA-EF1a-mCherry2_lenti
FEATURES             Location/Qualifiers
     source          1..8101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        232..611
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        613..811
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     766..786
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             829..1009
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1056..1181
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1674..1907
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2092..2136
                     /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    2434..2551
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2602..2850
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2602..2622
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2773..2792
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2877..2952
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        2996..4173
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3227..4164
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4121..4141
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4191..4200
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    4923..5511
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4976..4996)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5394..5405)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5583..5816
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5842..5859)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5848..6072
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6178..6195)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6349..6937)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6429..6448)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7108..7899,7900..7968))
                     /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     7731..7750
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7969..8073)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat tgacattgat
      241 tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
      301 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      361 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      421 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      481 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      541 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      601 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      661 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      721 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      781 ggcgtgtacg gtgggaggtc tatataagca gcgcgttttg cctgtactgg gtctctctgg
      841 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      901 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      961 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
     1021 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
     1081 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
     1141 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
     1201 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
     1261 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
     1321 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
     1381 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1441 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1501 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1561 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1621 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1681 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1741 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1801 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1861 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1921 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1981 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     2041 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     2101 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     2161 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     2221 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     2281 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt
     2341 ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact gcgtgcgcca
     2401 attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     2461 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     2521 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     2581 agtttggtta attaaggtac cgagggccta tttcccatga ttccttcata tttgcatata
     2641 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
     2701 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
     2761 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
     2821 ttatatatct tgtggaaagg acgaaacacc ggagacggag gacgacgaac gtctctgtta
     2881 tagtactctg gaaacagaat ctactataac aaggcaaaat gccgtgttta tctcgtcaac
     2941 ttgttggcga gatttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc
     3001 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
     3061 ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
     3121 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
     3181 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg
     3241 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc
     3301 cactggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt
     3361 tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg
     3421 cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat
     3481 aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat
     3541 agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc
     3601 ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg
     3661 ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc
     3721 gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg
     3781 tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg
     3841 cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca
     3901 gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc
     3961 tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt
     4021 ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc
     4081 ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt
     4141 caaagttttt ttcttccatt tcaggtgtcg tgaaccggtt ctagagcgct gccaccatgg
     4201 tcagtaaagg tgaagaggac aatatggcta tcatcaagga gttcatgcgc tttaaggtac
     4261 acatggaagg ctcagttaac gggcatgagt tcgaaataga gggagaaggt gaaggacgcc
     4321 cctatgaagg cacccaaacg gctaagctga aggtaactaa gggcggtccc ttgcccttcg
     4381 cctgggacat actgagcccg cagttcatgt acggttccaa agcctatgta aaacaccccg
     4441 ccgacattcc tgactatctt aagctgtctt ttcccgaagg ctttaactgg gaaagagtaa
     4501 tgaatttcga ggatggaggg gtggtcactg taacacagga ttctagtctg caagatgggg
     4561 agttcatcta caaggtgaag ctgcgcggca cgaattttcc ctcagatggt cctgtcatgc
     4621 agtgtcggac tatggggtgg gaagcaagca cagaaagaat gtatcctgag gacggggcac
     4681 tgaagggaga gatcaaacag cggctcaaac ttaaagacgg cgggcactat gacgccgaag
     4741 ttaagacaac ttacaaggcc aagaagcccg ttcagttgcc gggcgcatac aacgtggaca
     4801 taaagctcga catcttgtcc cataatgagg actacacaat cgtggaacag tatgaacggg
     4861 ccgagggcag acattctacc ggggggatgg atgagcttta caagtaaacg cgttaagtcg
     4921 acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     4981 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     5041 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     5101 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     5161 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     5221 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     5281 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     5341 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     5401 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     5461 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cgtcgacttt
     5521 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg
     5581 actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt gtactgggtc
     5641 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     5701 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     5761 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagggc
     5821 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     5881 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     5941 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     6001 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     6061 tgggctctat ggcttctgag gcggaaagaa ccagccatta atgaaaagaa ccagccatta
     6121 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     6181 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     6241 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     6301 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6361 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6421 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     6481 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     6541 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     6601 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6661 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6721 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6781 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6841 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6901 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6961 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     7021 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     7081 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     7141 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     7201 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     7261 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7321 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7381 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7441 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     7501 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7561 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7621 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7681 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7741 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7801 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7861 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7921 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7981 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     8041 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     8101 c
//
