LOCUS       sequence-422158-        4688 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Modular Unit carrying pegRNA and ngRNA scaffolds flanked by attL5 
            and attR3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4688)
  AUTHORS   Gupta A, Liu B, Raza S, Chen QJ, Yang B
  TITLE     Modularly assembled multiplex prime editors for simultaneous editing
            of agronomically important genes in rice.
  JOURNAL   Plant Commun. 2023 Oct 26:100741. doi: 10.1016/j.xplc.2023.100741.
  PUBMED    37897041
REFERENCE   2  (bases 1 to 4688)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant 
            Commun. 2023 Oct 26:100741. doi: 10.1016/j.xplc.2023.100741."
COMMENT     Sequence Label: pCR8-attL5-pegRNA-attR3
FEATURES             Location/Qualifiers
     source          1..4688
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      882..1470
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1371..1390
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1624..1641
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(1800..1827)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1919..2005)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2054..2076
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2068..2085
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2069..2085
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2101..2196
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     ncRNA           3443..3510
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     misc_RNA        3616..3691
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     ncRNA           3701..3768
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     terminator      3800..4048
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     protein_bind    4056..4179
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4215..4234)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4216..4234)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4239..4255)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4239..4255)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(4686..4688,1..789)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 agggaagcgg tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag
       61 cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc
      121 ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca
      181 acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag
      241 attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat
      301 ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc
      361 ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat
      421 agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat
      481 ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc
      541 gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa
      601 atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag
      661 cccgtcatac ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg
      721 cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc
      781 ggcaaataac cctcgagcca cccatgacca aaatccctta acgtgagtta cgcgtcgttc
      841 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
      901 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
      961 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1021 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1081 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1141 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1201 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1261 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1321 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1381 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     1441 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     1501 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     1561 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     1621 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     1681 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     1741 agtgagcgca acgcaattaa tacgcgtacc gctagccagg aagagtttgt agaaacgcaa
     1801 aaaggccatc cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc
     1861 gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat
     1921 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     1981 ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc gttaacgcta
     2041 gcatggatgt tttcccagtc acgacgttgt aaaacgacgg ccagtcttaa gctcgggccc
     2101 aaataatgat tttattttga ctgatagtga cctgttcgtt gcaacaaatt gatgagcaat
     2161 gcttttttat aatgccaact ttgtatacaa aagttgccga attatggagt caaagattca
     2221 aatagaggac ctaacagaac tcgccgtaaa gactggcgaa cagttcatac agagtctctt
     2281 acgactcaat gacaagaaga aaatcttcgt caacatggtg gagcacgaca cacttgtcta
     2341 ctccaaaaat atcaaagata cagtctcaga agaccaaagg gcaattgaga cttttcaaca
     2401 aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt
     2461 gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc
     2521 catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag
     2581 catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgattg
     2641 gcagacatac tgtcccacaa atgaagatgg aatctgtaaa agaaaacgcg tgaaataatg
     2701 cgtctgacaa aggttaggtc ggctgccttt aatcaatacc aaagtggtcc ctaccacgat
     2761 ggaaaaactg tgcagtcggt ttggcttttt ctgacgaaca aataagattc gtggccgaca
     2821 ggtgggggtc caccatgtga aggcatcttc agactccaat aatggagcaa tgacgtaagg
     2881 gcttacgaaa taagtaaggg tagtttggga aatgtccact cacccgtcag tctataaata
     2941 cttagcccct ccctcattgt taagggagca aaatctcaga gagatagtcc tagagagaga
     3001 aagagagcaa gtagcctaga agtagtcaag gcggcgaagt attcaggcac gtggccagga
     3061 agaagaaaag ccaagacgac gaaaacaggt aagagctaag catctagaaa gttgaaaaca
     3121 atcttcaaaa gtcccacatc gcttagataa gaaaacgaag ctgagtttat atacagctag
     3181 agtcgaagta gtgattgaac aaagcaccag tggtctagtg gtagaatagt accctgccac
     3241 ggtacagacc cgggttcgat tcccggctgg tgcaagagac gagtcacgtc tcagtttaag
     3301 agctatgctg gaaacagcat agcaagttta aataaggcta gtccgttatc aacttgaaaa
     3361 agtggcaccg agtcggtgca gagaccacag gatccttggt ctcacgcggt tctatctagt
     3421 tacgcgttaa accaactaga aaggccggca tggtcccagc ctcctcgctg gcgccggctg
     3481 ggcaacatgc ttcggcatgg cgaatgggac aacaacaaat cagagtggcg cagcggaagc
     3541 gtggtgggcc cataacccac aggtcccagg atcgaaacct ggctctgata agagaccaca
     3601 ggatccttgg tctcagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     3661 tcaacttgaa aaagtggcac cgagtcggtg cttttttttt ggccggcatg gtcccagcct
     3721 cctcgctggc gccggctggg caacatgctt cggcatggcg aatgggactt tttttttgat
     3781 atctccgggg ctaattgaat atgaagatga agatgaaata tttggtgtgt caaataaaaa
     3841 gctggtgtgc ttaagtttgt gtttttttct tggcttgttg tgttatgaat ttgtggcttt
     3901 ttctaatatt aaatgaatgt aagatctcat tataatgaat aaacaaatgt ttctataatc
     3961 cattgtgaat gttttgttgg atctcttctg cagcatataa ctactgtatg tgctatggta
     4021 tggactatgg aatatgatta aagataagga attcgcaact ttgtataata aagttgaacg
     4081 agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata aaaaacagac
     4141 tacataatac tgtaaaacac aacatatcca gtcactatga aaataaaatc attatttgcc
     4201 atccagctga tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc
     4261 tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat
     4321 gcctcctcta gaccagccag gacagaaatg cctcgacttc gctgctaccc aaggttgccg
     4381 ggtgacgcac accgtggaaa cggatgaagg cacgaaccca gtggacataa gcctgttcgg
     4441 ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga accttgaccg
     4501 aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac tgtttttttg
     4561 gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt
     4621 tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa acaaagttaa
     4681 acattatg
//
