LOCUS       sequence-370605-        4800 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Marathon-RT tetramutant (D14R-D74R-N116K-N197R).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4800)
  AUTHORS   Grunewald J, Miller BR, Szalay RN, Cabeceiras PK, Woodilla CJ, Holtz
            EJB, Petri K, Joung JK
  TITLE     Engineered CRISPR prime editors with compact, untethered reverse 
            transcriptases.
  JOURNAL   Nat Biotechnol. 2023 Mar;41(3):337-343. doi: 
            10.1038/s41587-022-01473-1. Epub 2022 Sep 26.
  PUBMED    36163548
REFERENCE   2  (bases 1 to 4800)
  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"; doi: 
            "10.1038/s41587-022-01473-1"; journalName: "Nat Biotechnol"; date: 
            "2023-03"; volume: "41"; issue: "3"; pages: "337-343"
COMMENT     Sequence Label: Marathon-RT tetramutant for Split-PE (pEH101)
FEATURES             Location/Qualifiers
     source          1..4800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        374..577
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     528..548
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     619..638
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        619..637
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             687..707
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2028..2048
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2057..2074
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2097..2114)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2103..2327
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2398..2414)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2398..2414)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2411..2433)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2422..2438
                     /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(2446..2452,2453..2470,2471..2476))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2491..2512
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2629..2646)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2800..3388)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2880..2899)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3559..4350,4351..4419))
                     /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     4182..4201
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4420..4524)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4603..4622)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(4794..4800,1..373)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
       61 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      121 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      181 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
      241 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      301 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      361 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      421 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      481 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      541 taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg
      601 ctagagatcc gcggccgcta atacgactca ctatagggag agccgccacc atgaaacgga
      661 cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcgac accagcaatc
      721 tgatggaaca gatcctgagc agccggaacc tgaaccgggc ctacctgcag gtggtgagaa
      781 ataaaggcgc tgaaggcgtt gatggcatga agtacaccga gctgaaggag catctggcca
      841 agaacggcga gacaatcaag ggccagctga gaaccagaaa gtataagcct cagccagcta
      901 gacgggtgga aatccccaag ccccggggcg gagtgcggaa cctgggagtg ccaacagtca
      961 cagaccggtt catccagcag gctatcgccc aagtgctgac ccctatctac gaggaacagt
     1021 ttcacgacca ctcttacggc ttccggccca agagatgcgc ccagcaagcc atcctgacag
     1081 ccctgaacat catgaacgat ggtaatgact ggatcgtgga catcgacctg gaaaagtttt
     1141 tcgataccgt gaatcacgat aagctgatga cgctgattgg cagaaccatc aaggacggcg
     1201 acgtgatctc tattgtgcgc aagtacctcg tgtccggcat catgatcgat gacgagtacg
     1261 aagatagcat cgtgggaaca cctcagggcg gccggctgtc tcctctgctg gccaacatca
     1321 tgctgaacga gctggataag gagatggaaa aaaggggcct gaacttcgtg cggtacgccg
     1381 acgactgcat catcatggtc ggctccgaga tgagcgccaa cagagtcatg cggaacatca
     1441 gcagattcat cgaagagaag ctgggcctga aagtgaacat gaccaagtcc aaggtggaca
     1501 gacctagcgg actgaagtac ttgggctttg gcttctactt cgaccccaga gcccaccagt
     1561 tcaaggccaa gcctcacgcc aagagcgtgg ctaagttcaa aaagagaatg aaagagctga
     1621 cctgtagaag ctggggcgtg tctaacagct acaaggtgga aaaactgaat caactgatca
     1681 gaggctggat caactacttc aagatcggca gcatgaagac cctgtgtaaa gagctggaca
     1741 gcagaatcag gtacagactg cggatgtgca tctggaagca gtggaaaacc cctcagaacc
     1801 aggagaaaaa cctggtcaag cttggaattg acagaaatac cgccagaaga gtggcctata
     1861 caggcaagcg aatcgcctac gtgtgcaaca agggcgccgt gaacgtggct atcagcaaca
     1921 agcggctggc cagcttcggc ctgatctcta tgctggacta ctacatcgag aagtgcgtga
     1981 cctgctctgg cggctcaaaa agaaccgccg acggcagcga attcgagccc aagaagaaga
     2041 ggaaagtcta accggtcatc atcaccatca ccattgagtt taaacccgct gatcagcctc
     2101 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     2161 cctggaaggt gccactccca ctgtcctttc ctaataaaat gagaaaattg catcgcattg
     2221 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     2281 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     2341 aagaaccagc tggggctcga taccgtcgac ctctagctag agcttggcgt aatcatggtc
     2401 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     2461 aagcataaag tgtaaagcct agggtgccta atgagtgagc taactcacat taattgcgtt
     2521 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     2581 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     2641 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2701 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     2761 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2821 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2881 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2941 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3001 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3061 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3121 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3181 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     3241 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3301 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3361 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3421 cactcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3481 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3541 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3601 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3661 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3721 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3781 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3841 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3901 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3961 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     4021 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     4081 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     4141 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     4201 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     4261 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4321 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4381 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     4441 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     4501 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg
     4561 atcgggagat cgatctcccg atcccctagg gtctactctc agtacaatct gctctgatgc
     4621 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     4681 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     4741 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
//
