LOCUS       sequence-370600-        5553 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MMLV-RT (full-length, from PE2) for Split-PE use.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5553)
  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 5553)
  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 for Split-PE (pEH45)
FEATURES             Location/Qualifiers
     source          1..5553
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        6..385
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..589
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     540..560
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     631..650
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        631..649
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             699..719
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2793..2813
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2822..2839
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2862..2879)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2868..3092
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3163..3179)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3163..3179)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3176..3198)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3187..3203
                     /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(3211..3217,3218..3235,3236..3241))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3256..3277
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3394..3411)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3565..4153)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3645..3664)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4324..5115,5116..5184))
                     /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     4947..4966
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5185..5289)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5368..5387)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
       61 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      121 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      181 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      241 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      301 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      361 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
      421 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
      481 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
      541 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa
      601 ccgtcagatc cgctagagat ccgcggccgc taatacgact cactataggg agagccgcca
      661 ccatgaaacg gacagccgac ggaagcgagt tcgagtcacc aaagaagaag cggaaagtca
      721 ccctaaatat agaagatgag tatcggctac atgagacctc aaaagagcca gatgtttctc
      781 tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac
      841 tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc cccgtgtcca
      901 taaaacaata ccccatgtca caagaagcca gactggggat caagccccac atacagagac
      961 tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc ctgctacccg
     1021 ttaagaaacc agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc
     1081 gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac
     1141 cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc ctgagactcc
     1201 accccaccag tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag
     1261 gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc ctgtttaatg
     1321 aggcactgca cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac
     1381 agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc
     1441 gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa
     1501 tttgccagaa acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga
     1561 ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct cgacaactaa
     1621 gggagttcct agggaaggca ggcttctgtc gcctcttcat ccctgggttt gcagaaatgg
     1681 cagcccccct gtaccctctc accaaaccgg ggactctgtt taattggggc ccagaccaac
     1741 aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg gggttgccag
     1801 atttgactaa gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc
     1861 taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa aagctagacc
     1921 cagtagcagc tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa
     1981 aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag
     2041 aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg actcactatc
     2101 aggccttgct tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg
     2161 ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat atcctggccg
     2221 aagcccacgg aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct
     2281 ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga
     2341 ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc gctcagcggg
     2401 ctgaactgat agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt
     2461 atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata tacagaaggc
     2521 gtgggtggct cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac
     2581 taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg
     2641 gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga aaggcagcca
     2701 tcacagagac tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tctggcggct
     2761 caaaaagaac cgccgacggc agcgaattcg agcccaagaa gaagaggaaa gtctaaccgg
     2821 tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg tgccttctag
     2881 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     2941 tcccactgtc ctttcctaat aaaatgagaa aattgcatcg cattgtctga gtaggtgtca
     3001 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     3061 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     3121 ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     3181 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3241 agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3301 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3361 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3421 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3481 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3541 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3601 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3661 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3721 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3781 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3841 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3901 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3961 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4021 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4081 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4141 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacactc agtggaacga
     4201 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4261 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4321 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4381 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4441 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4501 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4561 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4621 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4681 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4741 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4801 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4861 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4921 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4981 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5041 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5101 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5161 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5221 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5281 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc
     5341 tcccgatccc ctagggtcta ctctcagtac aatctgctct gatgccgcat agttaagcca
     5401 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     5461 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     5521 tgcgctgctt cgcgatgtac gggccagata tac
//
