LOCUS       sequence-370601-        5010 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MMLV-RT (from PE2) with truncated RNAse H domain (dRH) for
            Split-PE use.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5010)
  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 5010)
  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: MMLV-RT(dRH) for Split-PE (pEH50)
FEATURES             Location/Qualifiers
     source          1..5010
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..587
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     538..558
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             697..717
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2248..2268
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2277..2294
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2317..2334)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2323..2547
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2618..2634)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2618..2634)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2631..2653)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2642..2658
                     /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(2666..2672,2673..2690,2691..2696))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2711..2732
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2849..2866)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3020..3608)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3100..3119)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3779..4570,4571..4639))
                     /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     4402..4421
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4640..4744)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4823..4842)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc
      601 gtcagatccg ctagagatcc gcggccgcta atacgactca ctatagggag agccgccacc
      661 atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcacc
      721 ctaaatatag aagatgagta tcggctacat gagacctcaa aagagccaga tgtttctcta
      781 gggtccacat ggctgtctga ttttcctcag gcctgggcgg aaaccggggg catgggactg
      841 gcagttcgcc aagctcctct gatcatacct ctgaaagcaa cctctacccc cgtgtccata
      901 aaacaatacc ccatgtcaca agaagccaga ctggggatca agccccacat acagagactg
      961 ttggaccagg gaatactggt accctgccag tccccctgga acacgcccct gctacccgtt
     1021 aagaaaccag ggactaatga ttataggcct gtccaggatc tgagagaagt caacaagcgg
     1081 gtggaagaca tccaccccac cgtgcccaac ccttacaacc tcttgagcgg gctcccaccg
     1141 tcccaccagt ggtacactgt gcttgattta aaggatgcct ttttctgcct gagactccac
     1201 cccaccagtc agcctctctt cgcctttgag tggagagatc cagagatggg aatctcagga
     1261 caattgacct ggaccagact cccacagggt ttcaaaaaca gtcccaccct gtttaatgag
     1321 gcactgcaca gagacctagc agacttccgg atccagcacc cagacttgat cctgctacag
     1381 tacgtggatg acttactgct ggccgccact tctgagctag actgccaaca aggtactcgg
     1441 gccctgttac aaaccctagg gaacctcggg tatcgggcct cggccaagaa agcccaaatt
     1501 tgccagaaac aggtcaagta tctggggtat cttctaaaag agggtcagag atggctgact
     1561 gaggccagaa aagagactgt gatggggcag cctactccga agacccctcg acaactaagg
     1621 gagttcctag ggaaggcagg cttctgtcgc ctcttcatcc ctgggtttgc agaaatggca
     1681 gcccccctgt accctctcac caaaccgggg actctgttta attggggccc agaccaacaa
     1741 aaggcctatc aagaaatcaa gcaagctctt ctaactgccc cagccctggg gttgccagat
     1801 ttgactaagc cctttgaact ctttgtcgac gagaagcagg gctacgccaa aggtgtccta
     1861 acgcaaaaac tgggaccttg gcgtcggccg gtggcctacc tgtccaaaaa gctagaccca
     1921 gtagcagctg ggtggccccc ttgcctacgg atggtagcag ccattgccgt actgacaaag
     1981 gatgcaggca agctaaccat gggacagcca ctagtcattc tggcccccca tgcagtagag
     2041 gcactagtca aacaaccccc cgaccgctgg ctttccaacg cccggatgac tcactatcag
     2101 gccttgcttt tggacacgga ccgggtccag ttcggaccgg tggtagccct gaacccggct
     2161 acgctgctcc cactgcctga ggaagggctg caacacaact gcctttctgg cggctcaaaa
     2221 agaaccgccg acggcagcga attcgagccc aagaagaaga ggaaagtcta accggtcatc
     2281 atcaccatca ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     2341 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2401 ctgtcctttc ctaataaaat gagaaaattg catcgcattg tctgagtagg tgtcattcta
     2461 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     2521 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga
     2581 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2641 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2701 agggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2761 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2821 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2881 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2941 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     3001 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     3061 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     3121 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     3181 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     3241 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3301 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3361 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3421 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3481 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3541 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3601 gatctcaaga agatcctttg atcttttcta cggggtctga cactcagtgg aacgaaaact
     3661 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3721 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3781 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3841 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3901 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3961 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4021 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4081 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4141 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4201 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4261 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4321 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4381 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4441 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4501 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4561 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4621 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4681 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4741 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg
     4801 atcccctagg gtctactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     4861 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     4921 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     4981 tgcttcgcga tgtacgggcc agatatacgc
//
