LOCUS       sequence-275084-        8111 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lenti expression of mCherry2 with U6 promoter for SpyCas9 sgRNA 
            targeting GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8111)
  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 8111)
  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: pCF820_U6-sgRNA-EF1a-mCherry2_lenti
FEATURES             Location/Qualifiers
     source          1..8111
                     /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"
     promoter        3006..4183
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3237..4174
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4131..4151
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      4201..4210
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    4933..5521
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4986..5006)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5404..5415)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5593..5826
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5852..5869)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5858..6082
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6188..6205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6359..6947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6439..6458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7118..7909,7910..7978))
                     /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     7741..7760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7979..8083)
                     /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 gtctctgttt
     2881 aagagctatg ctggaaacag catagcaagt ttaaataagg ctagtccgtt atcaacttga
     2941 aaaagtggca ccgagtcggt gctttttttg aattcgctag ctaggtcttg aaaggagtgg
     3001 gaattggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
     3061 tggggggagg ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg
     3121 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     3181 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa
     3241 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt
     3301 gaattacttc cactggctgc agtacgtgat tcttgatccc gagcttcggg ttggaagtgg
     3361 gtgggagagt tcgaggcctt gcgcttaagg agccccttcg cctcgtgctt gagttgaggc
     3421 ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc
     3481 tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt
     3541 ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt cggtttttgg
     3601 ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct
     3661 gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa gctggccggc ctgctctggt
     3721 gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc
     3781 accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg
     3841 gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga aaagggcctt
     3901 tccgtcctca gccgtcgctt catgtgactc cacggagtac cgggcgccgt ccaggcacct
     3961 cgattagttc tcgagctttt ggagtacgtc gtctttaggt tggggggagg ggttttatgc
     4021 gatggagttt ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat
     4081 gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca
     4141 gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaaccggtt ctagagcgct
     4201 gccaccatgg tcagtaaagg tgaagaggac aatatggcta tcatcaagga gttcatgcgc
     4261 tttaaggtac acatggaagg ctcagttaac gggcatgagt tcgaaataga gggagaaggt
     4321 gaaggacgcc cctatgaagg cacccaaacg gctaagctga aggtaactaa gggcggtccc
     4381 ttgcccttcg cctgggacat actgagcccg cagttcatgt acggttccaa agcctatgta
     4441 aaacaccccg ccgacattcc tgactatctt aagctgtctt ttcccgaagg ctttaactgg
     4501 gaaagagtaa tgaatttcga ggatggaggg gtggtcactg taacacagga ttctagtctg
     4561 caagatgggg agttcatcta caaggtgaag ctgcgcggca cgaattttcc ctcagatggt
     4621 cctgtcatgc agtgtcggac tatggggtgg gaagcaagca cagaaagaat gtatcctgag
     4681 gacggggcac tgaagggaga gatcaaacag cggctcaaac ttaaagacgg cgggcactat
     4741 gacgccgaag ttaagacaac ttacaaggcc aagaagcccg ttcagttgcc gggcgcatac
     4801 aacgtggaca taaagctcga catcttgtcc cataatgagg actacacaat cgtggaacag
     4861 tatgaacggg ccgagggcag acattctacc ggggggatgg atgagcttta caagtaaacg
     4921 cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     4981 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     5041 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     5101 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     5161 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     5221 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     5281 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     5341 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     5401 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     5461 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     5521 cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     5581 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
     5641 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     5701 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     5761 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     5821 ctagcagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     5881 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     5941 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     6001 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     6061 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagccatta atgaaaagaa
     6121 ccagccatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     6181 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     6241 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     6301 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     6361 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     6421 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     6481 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6541 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6601 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6661 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6721 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6781 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6841 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6901 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6961 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     7021 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     7081 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     7141 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     7201 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     7261 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     7321 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     7381 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     7441 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     7501 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7561 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7621 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7681 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7741 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7801 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7861 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7921 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7981 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     8041 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     8101 tgccacctga c
//
