LOCUS       sequence-464557-        6540 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows the Golden Gate (BsmBI) cloning of pegRNA between two 
            Csy4-hairpins in the intron of a CMV-FLUC encoding plasmid (POL II):
            ipegRNAs must be processed by Csy4 in VLP- producer cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6540)
  AUTHORS   Halegua T, Risson V, Carras J, Rouyer M, Coudert L, Jacquier A, 
            Schaeffer L, Ohlmann T, Mangeot PE
  TITLE     Delivery of Prime editing in human stem cells using pseudoviral 
            NanoScribes particles.
  JOURNAL   Nat Commun. 2025 Jan 4;16(1):397. doi: 10.1038/s41467-024-55604-0.
  PUBMED    39755699
REFERENCE   2  (bases 1 to 6540)
  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 
            Commun."; date: "2025-01-4"; volume: "16(1):397. doi"; pages: " 
            10.1038/s41467-024-55604-0"
COMMENT     Sequence Label: pp-CBBC-Fluc
FEATURES             Location/Qualifiers
     source          1..6540
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     52..72
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     98..122
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    747..766
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     protein_bind    791..810
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     primer_bind     864..883
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             917..2566
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     primer_bind     complement(983..1001)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     primer_bind     complement(2666..2685)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2731..2786
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2785..2804)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3144..3160)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3144..3160)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3157..3179)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3168..3184
                     /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)."
     promoter        complement(join(3192..3198,3199..3216,3217..3222))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3237..3258
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3375..3392)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3546..4134)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3626..3645)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4305..5096,5097..5165))
                     /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     4928..4947
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5166..5270)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5296..5751)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5433..5454)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5645..5664
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5878..5900
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5892..5909
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5893..5909
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5919..5938
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5919..5937
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     enhancer        6058..6437
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(6438..6540,1..101)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
       61 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga
      121 tcgcctggaa acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      181 gcctccggtc gaccgatcct gagaacttca gggtgagttt ggggaccctt gattgttctt
      241 tctttttcgc tattgtaaaa ttcatgttat atggaggggg caaagttttc agggtgttgt
      301 ttagaatggg aagatgtccc ttgtatcacc atggaccctc atgataattt tgtttctttc
      361 actttctact ctgttgacaa ccattgtctc ctcttatttt cttttcattt tcttgtaact
      421 ttttcgttaa actttagctt gcatttgtaa cgaattttta aattcacttt tgtttatttg
      481 tcagattgta agtactttct ctaatcactt ttttttcaag gcaatcaggg tatattatat
      541 tgtacttcag cacagtttta gagaacaatt gttataatta aatgataagg tagaatattt
      601 ctgcatataa attctggctg gcgtggaaat attcttattg gtagaaacaa ctacatcctg
      661 gtcatcatcc tgcctttctc tttatggtta caatgatata cactgtttga gatgaggata
      721 aaatactctg agtccaaacc gggcccgttc actgccgtat aggcaggaga cgacgcgtgg
      781 tacccgtctc gttcactgcc gtataggcag gggcccctct gctaaccatg ttcatgcctt
      841 cttctttttc ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg
      901 caaagaattc ctcgagatgg aagacgccaa aaacataaag aaaggcccgg cgccattcta
      961 tcctctagag gatggaaccg ctggagagca actgcataag gctatgaaga gatacgccct
     1021 ggttcctgga acaattgctt ttacagatgc acatatcgag gtgaacatca cgtacgcgga
     1081 atacttcgaa atgtccgttc ggttggcaga agctatgaaa cgatatgggc tgaatacaaa
     1141 tcacagaatc gtcgtatgca gtgaaaactc tcttcaattc tttatgccgg tgttgggcgc
     1201 gttatttatc ggagttgcag ttgcgcccgc gaacgacatt tataatgaac gtgaattgct
     1261 caacagtatg aacatttcgc agcctaccgt agtgtttgtt tccaaaaagg ggttgcaaaa
     1321 aattttgaac gtgcaaaaaa aattaccaat aatccagaaa attattatca tggattctaa
     1381 aacggattac cagggatttc agtcgatgta cacgttcgtc acatctcatc tacctcccgg
     1441 ttttaatgaa tacgattttg taccagagtc ctttgatcgt gacaaaacaa ttgcactgat
     1501 aatgaattcc tctggatcta ctgggttacc taagggtgtg gcccttccgc atagaactgc
     1561 ctgcgtcaga ttctcgcatg ccagagatcc tatttttggc aatcaaatca ttccggatac
     1621 tgcgatttta agtgttgttc cattccatca cggttttgga atgtttacta cactcggata
     1681 tttgatatgt ggatttcgag tcgtcttaat gtatagattt gaagaagagc tgtttttacg
     1741 atcccttcag gattacaaaa ttcaaagtgc gttgctagta ccaaccctat tttcattctt
     1801 cgccaaaagc actctgattg acaaatacga tttatctaat ttacacgaaa ttgcttctgg
     1861 gggcgcacct ctttcgaaag aagtcgggga agcggttgca aaacgcttcc atcttccagg
     1921 gatacgacaa ggatatgggc tcactgagac tacatcagct attctgatta cacccgaggg
     1981 ggatgataaa ccgggcgcgg tcggtaaagt tgttccattt tttgaagcga aggttgtgga
     2041 tctggatacc gggaaaacgc tgggcgttaa tcagagaggc gaattatgtg tcagaggacc
     2101 tatgattatg tccggttatg taaacaatcc ggaagcgacc aacgccttga ttgacaagga
     2161 tggatggcta cattctggag acatagctta ctgggacgaa gacgaacact tcttcatagt
     2221 tgaccgcttg aagtctttaa ttaaatacaa aggatatcag gtggcccccg ctgaattgga
     2281 atcgatattg ttacaacacc ccaacatctt cgacgcgggc gtggcaggtc ttcccgacga
     2341 tgacgccggt gaacttcccg ccgccgttgt tgttttggag cacggaaaga cgatgacgga
     2401 aaaagagatc gtggattacg tcgccagtca agtaacaacc gcgaaaaagt tgcgcggagg
     2461 agttgtgttt gtggacgaag taccgaaagg tcttaccgga aaactcgacg caagaaaaat
     2521 cagagagatc ctcataaagg ccaagaaggg cggaaagtcc aaattggcta ggatccgtcg
     2581 aggaattcac tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg
     2641 ccctggctca caaataccac tgagatcttt ttccctctgc caaaaattat ggggacatca
     2701 tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag
     2761 tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa
     2821 aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca
     2881 tgaacaaagg ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt
     2941 ccttattcca tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt
     3001 tatttttttc tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc
     3061 tcctctcctg actactccca gtcatagctg tccctcttct cttatggaga tccctcgacg
     3121 gatcggccgc aattcgtaat catgtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3181 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3241 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3301 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3361 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3421 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3481 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3541 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3601 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3661 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3721 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3781 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3841 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3901 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3961 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4021 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4081 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4141 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4201 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4261 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4321 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4381 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4441 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4501 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4561 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4621 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4681 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4741 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4801 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4861 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4921 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4981 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5041 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5101 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5161 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5221 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5281 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     5341 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     5401 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     5461 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     5521 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     5581 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     5641 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     5701 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     5761 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     5821 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     5881 agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg
     5941 aattggagct ccaccgcggt ggcggccgct ctagagagct tggcccattg catacgttgt
     6001 atccatatca taatatgtac atttatattg gctcatgtcc aacattaccg ccatgttgac
     6061 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     6121 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     6181 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     6241 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     6301 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     6361 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     6421 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     6481 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
//
