LOCUS       sequence-425129-        7611 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CRISPRa gRNA for CD40 promoter and mScarlet. gRNA driven 
            by mU6..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7611)
  AUTHORS   Pacalin NM, Steinhart Z, Shi Q, Belk JA, Dorovskyi D, Kraft K, 
            Parker KR, Shy BR, Marson A, Chang HY
  TITLE     Bidirectional epigenetic editing reveals hierarchies in gene 
            regulation.
  JOURNAL   Nat Biotechnol. 2024 May 17. doi: 10.1038/s41587-024-02213-3.
  PUBMED    38760566
REFERENCE   2  (bases 1 to 7611)
  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: "Nat 
            Biotechnol. 2024 May 17. doi: 10.1038/s41587-024-02213-3."
COMMENT     Sequence Label: p105_gRNA_Sa_CD40
FEATURES             Location/Qualifiers
     source          1..7611
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1211..1230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1402..1781
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1783..1981
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             1999..2179
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2226..2351
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2848..3081
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3266..3310
                     /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    3608..3725
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(3978..4011)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     4015..4035
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        4119..4194
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     enhancer        4319..4622
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4623..4826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             4860..5555
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(4993..5011)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5261..5280
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5575..5608)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5664..6252
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5717..5737)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6135..6146)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6255..6271)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6256..6272)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6781..6961
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7023..7611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7103..7122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     1201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     1261 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     1321 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     1381 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     1441 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1501 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1561 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1621 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1681 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1741 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1801 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1861 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1921 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1981 gcgcgttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     2041 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     2101 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     2161 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
     2221 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
     2281 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
     2341 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
     2401 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
     2461 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     2521 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     2581 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     2641 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccggccgct
     2701 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
     2761 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
     2821 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
     2881 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
     2941 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     3001 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     3061 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     3121 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     3181 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     3241 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     3301 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     3361 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     3421 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg
     3481 acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat
     3541 tcgattagtg aacggatcgg cactgcgtgc gccaattctg cagacaaatg gcagtattca
     3601 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     3661 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     3721 attttcgggt ttattacagg gacagcagag atccagtttg gttagtaccg ggcccgctct
     3781 agagatccga cgcgccatct ctaggcccgc gccggccccc tcgcacggac ttgtgggaga
     3841 agctcggcta ctcccctgcc ccggttaatt tgcatataat atttcctagt aactatagag
     3901 gcttaatgtg cgataaaaga cagataatct gttcttttta atactagcta cattttacat
     3961 gataggcttg gatttctata acttcgtata gcatacatta tacgaagtta taaacagcac
     4021 aaaaggaaac tcaccctaac tgtaaagtaa ttgtgtgttt tgagactata agtatccctt
     4081 ggagaaccac cttgttggat gcgtccctaa actcccgggt tatagtactc tggaaacaga
     4141 atctactata acaaggcaaa atgccgtgtt tatctcgtca acttgttggc gagatttttt
     4201 tctcgagtac taggatccat taggcggccg cgtggataac cgtattaccg ccatgcatta
     4261 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     4321 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     4381 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     4441 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
     4501 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
     4561 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
     4621 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
     4681 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
     4741 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
     4801 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcccacca
     4861 tggtgagcaa gggcgaggca gtgatcaagg agttcatgcg gttcaaggtg cacatggagg
     4921 gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     4981 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc tcctgggaca
     5041 tcctgtcccc tcagttcatg tacggctcca gggccttcac caagcacccc gccgacatcc
     5101 ccgactacta taagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     5161 aggacggcgg cgccgtgacc gtgacccagg acacctccct ggaggacggc accctgatct
     5221 acaaggtgaa gctccgcggc accaacttcc ctcctgacgg ccccgtaatg cagaagaaga
     5281 caatgggctg ggaagcgtcc accgagcggt tgtaccccga ggacggcgtg ctgaagggcg
     5341 acattaagat ggccctgcgc ctgaaggacg gcggccgcta cctggcggac ttcaagacca
     5401 cctacaaggc caagaagccc gtgcagatgc ccggcgccta caacgtcgac cgcaagttgg
     5461 acatcacctc ccacaacgag gactacaccg tggtggaaca gtacgaacgc tccgagggcc
     5521 gccactccac cggcggcatg gacgagctgt acaaggaatt cgtcgaggga cctaataact
     5581 tcgtatagca tacattatac gaagttatac atgtttaagg gttccggttc cactaggtac
     5641 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     5701 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     5761 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     5821 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     5881 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     5941 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     6001 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     6061 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     6121 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     6181 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     6241 ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca tggagcaatc
     6301 acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag
     6361 gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca
     6421 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     6481 caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat
     6541 tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg atggtgctac
     6601 aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga gaacacccgc
     6661 ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt attagagtgg
     6721 aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc ggactgtact
     6781 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     6841 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     6901 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     6961 agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     7021 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     7081 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     7141 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     7201 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     7261 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     7321 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     7381 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     7441 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     7501 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     7561 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca a
//
