LOCUS       sequence-357437-        5590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MMLV-RT (3XFLAG-SV40-MMLVRT).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5590)
  AUTHORS   Liu B, Dong X, Cheng H, Zheng C, Chen Z, Rodriguez TC, Liang SQ, Xue
            W, Sontheimer EJ
  TITLE     A split prime editor with untethered reverse transcriptase and 
            circular RNA template.
  JOURNAL   Nat Biotechnol. 2022 Apr 4. pii: 10.1038/s41587-022-01255-9. doi: 
            10.1038/s41587-022-01255-9.
  PUBMED    35379962
REFERENCE   2  (bases 1 to 5590)
  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"; journalName: "Nat 
            Biotechnol. 2022 Apr 4. pii: 10.1038/s41587-022-01255-9. doi: 
            10.1038/s41587-022-01255-9."
COMMENT     Sequence Label: MMLV-RT
FEATURES             Location/Qualifiers
     source          1..5590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      626..635
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             635..700
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             707..727
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2825..2845
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2854..2871
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2894..2911)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2900..3124
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3195..3211)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3195..3211)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3208..3230)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3219..3235
                     /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(3243..3249,3250..3267,3268..3273))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3288..3309
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3426..3443)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3597..4185)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3677..3696)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4356..5147,5148..5216))
                     /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     4979..4998
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5217..5321)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5400..5419)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gagatccgcg gccgcgccac catggactat aaggaccacg acggagacta
      661 caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg
      721 gaaggtcggt atccacggag tcccagcagc caccctaaat atagaagatg agtatcggct
      781 acatgagacc tcaaaagagc cagatgtttc tctagggtcc acatggctgt ctgattttcc
      841 tcaggcctgg gcggaaaccg ggggcatggg actggcagtt cgccaagctc ctctgatcat
      901 acctctgaaa gcaacctcta cccccgtgtc cataaaacaa taccccatgt cacaagaagc
      961 cagactgggg atcaagcccc acatacagag actgttggac cagggaatac tggtaccctg
     1021 ccagtccccc tggaacacgc ccctgctacc cgttaagaaa ccagggacta atgattatag
     1081 gcctgtccag gatctgagag aagtcaacaa gcgggtggaa gacatccacc ccaccgtgcc
     1141 caacccttac aacctcttga gcgggctccc accgtcccac cagtggtaca ctgtgcttga
     1201 tttaaaggat gcctttttct gcctgagact ccaccccacc agtcagcctc tcttcgcctt
     1261 tgagtggaga gatccagaga tgggaatctc aggacaattg acctggacca gactcccaca
     1321 gggtttcaaa aacagtccca ccctgtttaa tgaggcactg cacagagacc tagcagactt
     1381 ccggatccag cacccagact tgatcctgct acagtacgtg gatgacttac tgctggccgc
     1441 cacttctgag ctagactgcc aacaaggtac tcgggccctg ttacaaaccc tagggaacct
     1501 cgggtatcgg gcctcggcca agaaagccca aatttgccag aaacaggtca agtatctggg
     1561 gtatcttcta aaagagggtc agagatggct gactgaggcc agaaaagaga ctgtgatggg
     1621 gcagcctact ccgaagaccc ctcgacaact aagggagttc ctagggaagg caggcttctg
     1681 tcgcctcttc atccctgggt ttgcagaaat ggcagccccc ctgtaccctc tcaccaaacc
     1741 ggggactctg tttaattggg gcccagacca acaaaaggcc tatcaagaaa tcaagcaagc
     1801 tcttctaact gccccagccc tggggttgcc agatttgact aagccctttg aactctttgt
     1861 cgacgagaag cagggctacg ccaaaggtgt cctaacgcaa aaactgggac cttggcgtcg
     1921 gccggtggcc tacctgtcca aaaagctaga cccagtagca gctgggtggc ccccttgcct
     1981 acggatggta gcagccattg ccgtactgac aaaggatgca ggcaagctaa ccatgggaca
     2041 gccactagtc attctggccc cccatgcagt agaggcacta gtcaaacaac cccccgaccg
     2101 ctggctttcc aacgcccgga tgactcacta tcaggccttg cttttggaca cggaccgggt
     2161 ccagttcgga ccggtggtag ccctgaaccc ggctacgctg ctcccactgc ctgaggaagg
     2221 gctgcaacac aactgccttg atatcctggc cgaagcccac ggaacccgac ccgacctaac
     2281 ggaccagccg ctcccagacg ccgaccacac ctggtacacg gatggaagca gtctcttaca
     2341 agagggacag cgtaaggcgg gagctgcggt gaccaccgag accgaggtaa tctgggctaa
     2401 agccctgcca gccgggacat ccgctcagcg ggctgaactg atagcactca cccaggccct
     2461 aaagatggca gaaggtaaga agctaaatgt ttatactgat agccgttatg cttttgctac
     2521 tgcccatatc catggagaaa tatacagaag gcgtgggtgg ctcacatcag aaggcaaaga
     2581 gatcaaaaat aaagacgaga tcttggccct actaaaagcc ctctttctgc ccaaaagact
     2641 tagcataatc cattgtccag gacatcaaaa gggacacagc gccgaggcta gaggcaaccg
     2701 gatggctgac caagcggccc gaaaggcagc catcacagag actccagaca cctctaccct
     2761 cctcatagaa aattcatcac cctctggcgg ctcaaaaaga accgccgacg gcagcgaatt
     2821 cgagcccaag aagaagagga aagtctaacc ggtcatcatc accatcacca ttgagtttaa
     2881 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     2941 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     3001 aaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     3061 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     3121 atggcttctg aggcggaaag aaccagctgg ggctcgatac cgtcgacctc tagctagagc
     3181 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3241 cacaacatac gagccggaag cataaagtgt aaagcctagg gtgcctaatg agtgagctaa
     3301 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3361 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3421 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3481 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3541 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3601 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3661 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3721 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3781 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3841 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3901 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3961 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4021 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4081 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4141 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4201 ttttctacgg ggtctgacac tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4261 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4321 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4381 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4441 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4501 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4561 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4621 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4681 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4741 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4801 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4861 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4921 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4981 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5041 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5101 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5161 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5221 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5281 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5341 ccacctgacg tcgacggatc gggagatcga tctcccgatc ccctagggtc tactctcagt
     5401 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     5461 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     5521 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     5581 tatacgcgtt
//
