LOCUS       sequence-232092-        7283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminal Npu DnaE Intein-SaKKH-BE3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7283)
  AUTHORS   Villiger L, Grisch-Chan HM, Lindsay H, Ringnalda F, Pogliano CB, 
            Allegri G, Fingerhut R, Haberle J, Matos J, Robinson MD, Thony B, 
            Schwank G
  TITLE     Treatment of a metabolic liver disease by in vivo genome base 
            editing in adult mice.
  JOURNAL   Nat Med. 2018 Oct;24(10):1519-1525. doi: 10.1038/s41591-018-0209-1. 
            Epub 2018 Oct 8.
  PUBMED    30297904
REFERENCE   2  (bases 1 to 7283)
  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/s41591-018-0209-1"; journalName: "Nat Med"; date: 
            "2018-10"; volume: "24"; issue: "10"; pages: "1519-1525"
COMMENT     Sequence Label: pLV302
FEATURES             Location/Qualifiers
     source          1..7283
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     enhancer        178..557
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        558..761
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     712..732
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      780..789
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             792..812
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             822..845
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             870..1556
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     primer_bind     complement(4149..4166)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4155..4362
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4371..4511
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4371..4500
                     /label=AAV2 ITR
     rep_origin      4586..5041
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4673..4692)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4883..4904
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5058..5077)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5177..5199
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5237..5255)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5323..5427
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5428..5496,5497..6288)
                     /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     complement(5646..5665)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6459..7047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6948..6967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctactcg aggcgttgac
      181 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      241 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      301 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      361 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      421 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      481 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      541 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      601 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      661 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      721 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actaccggtg
      781 ccaccatggg acccaagaag aagaggaaag tcgccgcagc ggactacaag gatgatgatg
      841 ataaaggtat tcacggggtc ccagccgcga tgagctcaga gactggccca gtggctgtgg
      901 accccacatt gagacggcgg atcgagcccc atgagtttga ggtattcttc gatccgagag
      961 agctccgcaa ggagacctgc ctgctttacg aaattaattg ggggggccgg cactccattt
     1021 ggcgacatac atcacagaac actaacaagc acgtcgaagt caacttcatc gagaagttca
     1081 cgacagaaag atatttctgt ccgaacacaa ggtgcagcat tacctggttt ctcagctgga
     1141 gcccatgcgg cgaatgtagt agggccatca ctgaattcct gtcaaggtat ccccacgtca
     1201 ctctgtttat ttacatcgca aggctgtacc accacgctga cccccgcaat cgacaaggcc
     1261 tgcgggattt gatctcttca ggtgtgacta tccaaattat gactgagcag gagtcaggat
     1321 actgctggag aaactttgtg aattatagcc cgagtaatga agcccactgg cctaggtatc
     1381 cccatctgtg ggtacgactg tacgttcttg aactgtactg catcatactg ggcctgcctc
     1441 cttgtctcaa cattctgaga aggaagcagc cacagctgac attctttacc atcgctcttc
     1501 agtcttgtca ttaccagcga ctgcccccac acattctctg ggccaccggg ttgaaaagcg
     1561 gcagcgagac tcccgggacc tcagagtccg ccacacccga aagtgggaaa cggaactaca
     1621 tcctggggct tgccattggg ataaccagcg ttggctacgg aattattgat tatgagacac
     1681 gcgatgtgat tgacgccggg gttaggctgt tcaaagaggc caacgttgaa aacaacgagg
     1741 gaagacggag taagcgcgga gcaagaagac tcaagcgcag acggagacat cggattcaga
     1801 gggtgaaaaa gctgctcttc gattacaatc tcctgaccga tcatagtgag ctgagcggaa
     1861 tcaaccccta cgaggcgcga gtgaaagggc tttcccagaa gctgtccgaa gaggagttct
     1921 ccgccgcgtt gctgcacctg gccaaacgga ggggggttca caatgtaaac gaagtggagg
     1981 aggacacggg caatgaactt agtacgaaag aacagatcag taggaactct aaggctctcg
     2041 aagagaaata cgtcgctgag ttgcagcttg agagactgaa aaaagacggc gaagtacgcg
     2101 gatctattaa taggttcaag acttcagatt acgtaaagga agccaagcag ctcctgaaag
     2161 tacagaaagc gtaccatcag ctcgatcaga gcttcatcga tacctacata gatttgctgg
     2221 agacacggag gacatactac gagggcccag gggaaggatc tccttttggg tggaaggaca
     2281 tcaaggaatg gtacgagatg cttatgggac attgtacata ttttccggag gagctcagga
     2341 gcgtcaagta cgcctacaat gccgacctgt acaatgccct caatgacctc aataacctcg
     2401 tgattaccag ggacgagaac gagaagctgg agtactatga aaagttccag attatcgaga
     2461 atgtgtttaa gcagaagaag aagccgacac ttaagcagat tgcaaaggaa atcctcgtga
     2521 atgaggaaga tatcaaggga tacagagtga caagtacagg caagcccgag ttcacaaatc
     2581 tgaaggtgta ccacgatatt aaggacataa ccgcacgaaa ggagataatc gaaaacgctg
     2641 agctcctcga tcagatcgca aaaattctta ccatctacca gtctagtgag gacattcagg
     2701 aggaactgac taatctgaac agtgagctca cccaagagga aattgagcag atttcaaacc
     2761 tgaaaggcta caccgggacg cacaatctga gcctcaaagc aatcaacctc attctggatg
     2821 aactttggca cacaaatgac aaccaaattg ccatattcaa ccgcctgaaa ctggtgccaa
     2881 aaaaagtgga tctgtcacag caaaaggaaa tccctacaac cttggttgac gattttattc
     2941 tgtcccccgt tgtcaagcgg agcttcatcc agtcaatcaa ggtgatcaat gccatcatta
     3001 aaaaatacgg attgccaaac gatataatta tcgagcttgc acgagagaag aactcaaagg
     3061 acgcccagaa gatgattaac gaaatgcaga agcgcaaccg ccagacaaac gaacgcatag
     3121 aggaaattat aagaacaacc ggcaaagaga atgccaagta tctgatcgag aaaatcaagc
     3181 tgcacgacat gcaagaaggc aagtgcctgt actctctgga agctatccca ctcgaagatc
     3241 tgctgaataa tccattcaat tacgaggtgg accacatcat ccctagatcc gtaagctttg
     3301 acaattcctt caataacaaa gttctggtta aacaggagga aaattctaaa aaagggaacc
     3361 ggaccccgtt ccagtacctg agctccagtg acagcaagat tagctacgag acttttaaga
     3421 aacatattct gaatctggcc aaaggcaaag gcaggatcag caagaccaag aaggagtacc
     3481 tcctcgaaga acgcgacatt aacagattta gtgtgcagaa agatttcatc aaccgaaacc
     3541 ttgtcgatac tcggtacgcc acgagaggcc tgatgaatct cctcaggagc tacttccgcg
     3601 tcaataatct ggacgttaaa gtcaagagca taaatggggg attcaccagc tttctgagga
     3661 gaaagtggaa gtttaagaag gaacgaaaca aaggatacaa gcaccatgct gaggatgctt
     3721 tgatcatcgc taacgcggac tttatcttta aggaatggaa aaagctggat aaggcaaaga
     3781 aagtgatgga aaaccagatg ttcgaggaga agcaggcaga gtgtcttagc tacgagacag
     3841 agattcttac agtggagtat ggacttcttc ccatcggtaa aattgttgaa aagaggatcg
     3901 agtgcacggt ctacagtgtc gataataatg gtaacatata tacgcaaccc gtcgcgcagt
     3961 ggcatgatag aggcgagcag gaagtatttg agtattgtct ggaggatgga tctctgattc
     4021 gcgcgactaa ggaccataag tttatgacgg ttgacggaca aatgctccca atagacgaga
     4081 tatttgaacg agaactggat cttatgagag tcgataactt gccgaattaa ctagagctcg
     4141 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     4201 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     4261 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     4321 caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc aggaacccct
     4381 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     4441 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     4501 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4561 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4621 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     4681 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4741 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4801 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4861 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     4921 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     4981 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5041 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5101 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5161 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5221 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5281 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5341 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5401 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5461 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5521 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5581 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5641 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5701 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5761 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5821 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5881 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5941 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6001 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6061 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6121 aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt
     6181 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6241 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6301 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6361 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6421 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6481 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6541 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6601 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6661 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6721 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6781 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6841 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6901 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6961 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7021 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7081 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     7141 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     7201 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     7261 cgttggccga ttcattaatg cag
//
