LOCUS       sequence-370604-        4800 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Marathon-RT (human codon optimization).
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 for Split-PE (pEH59)
FEATURES             Location/Qualifiers
     source          1..4800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        10..389
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        390..593
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     544..564
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     635..654
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        635..653
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             703..723
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2044..2064
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2073..2090
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2113..2130)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2119..2343
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2414..2430)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2414..2430)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2427..2449)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2438..2454
                     /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(2462..2468,2469..2486,2487..2492))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2507..2528
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2645..2662)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2816..3404)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2896..2915)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3575..4366,4367..4435))
                     /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     4198..4217
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4436..4540)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4619..4638)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
       61 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      121 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      181 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      241 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      301 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      361 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      421 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      481 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      541 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
      601 tgaaccgtca gatccgctag agatccgcgg ccgctaatac gactcactat agggagagcc
      661 gccaccatga aacggacagc cgacggaagc gagttcgagt caccaaagaa gaagcggaaa
      721 gtcgacacca gcaatctgat ggaacagatc ctgagcagcg acaacctgaa ccgggcctac
      781 ctgcaggtgg tgagaaataa aggcgctgaa ggcgttgatg gcatgaagta caccgagctg
      841 aaggagcatc tggccaagaa cggcgagaca atcaagggcc agctgagaac cagaaagtat
      901 aagcctcagc cagctagacg ggtggaaatc cccaagcccg atggcggagt gcggaacctg
      961 ggagtgccaa cagtcacaga ccggttcatc cagcaggcta tcgcccaagt gctgacccct
     1021 atctacgagg aacagtttca cgaccactct tacggcttcc ggcccaacag atgcgcccag
     1081 caagccatcc tgacagccct gaacatcatg aacgatggta atgactggat cgtggacatc
     1141 gacctggaaa agtttttcga taccgtgaat cacgataagc tgatgacgct gattggcaga
     1201 accatcaagg acggcgacgt gatctctatt gtgcgcaagt acctcgtgtc cggcatcatg
     1261 atcgatgacg agtacgaaga tagcatcgtg ggaacacctc agggcggcaa cctgtctcct
     1321 ctgctggcca acatcatgct gaacgagctg gataaggaga tggaaaaaag gggcctgaac
     1381 ttcgtgcggt acgccgacga ctgcatcatc atggtcggct ccgagatgag cgccaacaga
     1441 gtcatgcgga acatcagcag attcatcgaa gagaagctgg gcctgaaagt gaacatgacc
     1501 aagtccaagg tggacagacc tagcggactg aagtacttgg gctttggctt ctacttcgac
     1561 cccagagccc accagttcaa ggccaagcct cacgccaaga gcgtggctaa gttcaaaaag
     1621 agaatgaaag agctgacctg tagaagctgg ggcgtgtcta acagctacaa ggtggaaaaa
     1681 ctgaatcaac tgatcagagg ctggatcaac tacttcaaga tcggcagcat gaagaccctg
     1741 tgtaaagagc tggacagcag aatcaggtac agactgcgga tgtgcatctg gaagcagtgg
     1801 aaaacccctc agaaccagga gaaaaacctg gtcaagcttg gaattgacag aaataccgcc
     1861 agaagagtgg cctatacagg caagcgaatc gcctacgtgt gcaacaaggg cgccgtgaac
     1921 gtggctatca gcaacaagcg gctggccagc ttcggcctga tctctatgct ggactactac
     1981 atcgagaagt gcgtgacctg ctctggcggc tcaaaaagaa ccgccgacgg cagcgaattc
     2041 gagcccaaga agaagaggaa agtctaaccg gtcatcatca ccatcaccat tgagtttaaa
     2101 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2161 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgaga
     2221 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2281 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     2341 tggcttctga ggcggaaaga accagctggg gctcgatacc gtcgacctct agctagagct
     2401 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     2461 acaacatacg agccggaagc ataaagtgta aagcctaggg tgcctaatga gtgagctaac
     2521 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     2581 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     2641 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2701 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2761 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2821 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2881 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2941 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     3001 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     3061 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     3121 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     3181 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     3241 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     3301 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     3361 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     3421 tttctacggg gtctgacact cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     3481 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     3541 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     3601 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     3661 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     3721 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3781 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3841 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3901 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3961 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4021 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4081 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4141 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4201 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4261 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4321 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     4381 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     4441 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4501 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4561 cacctgacgt cgacggatcg ggagatcgat ctcccgatcc cctagggtct actctcagta
     4621 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     4681 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     4741 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
//
