LOCUS       sequence-232093-        6516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses C-terminal Npu DnaE Intein-SaKKH-BE3, tagRFP, and sgRNA in
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6516)
  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 6516)
  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: pLV312.3
FEATURES             Location/Qualifiers
     source          1..6516
                     /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"
     CDS             1839..1859
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1866..1931
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1944..2192
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             2205..2225
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2235..2291
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2292..3005
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     primer_bind     complement(3024..3041)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3030..3237
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3244..3492
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3244..3264
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3415..3434
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3515..3590
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   3604..3744
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3604..3733
                     /label=AAV2 ITR
     rep_origin      3819..4274
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3906..3925)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4116..4137
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4291..4310)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4410..4432
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4470..4488)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4556..4660
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4661..4729,4730..5521)
                     /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(4879..4898)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5692..6280
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6181..6200
                     /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 ccaccatgat caaaattgct actaggaagt atttgggaaa gcagaacgtc tatgacatcg
      841 gagtagaacg agaccacaac tttgccttga agaacggttt tatcgcaagc tcaatgcctg
      901 agatcgagac agagcaggaa tacaaggaaa ttttcatcac ccctcatcag attaaacaca
      961 taaaggactt caaagactat aaatactctc atagggtgga caaaaaaccc aatcgcaagc
     1021 tcattaatga caccctgtac tcaacacgga aggatgataa aggtaatacc ttgattgtga
     1081 ataatcttaa tggattgtat gacaaagata acgacaagct caagaagctg atcaacaagt
     1141 ctccagagaa gctccttatg tatcaccacg acccacagac ttatcagaaa ttgaaactga
     1201 tcatggagca atacggggat gagaagaacc cactctacaa atattatgag gaaacaggta
     1261 attacctgac caagtactcc aagaaggata acggaccagt gatcaaaaag ataaagtact
     1321 atggcaacaa acttaatgcg catttggaca taactgacga ttaccccaat tctcgaaaca
     1381 aggttgtgaa gctctccctg aagccttata gatttgacgt gtacctggat aatggggttt
     1441 ataaattcgt caccgtgaaa aatctggacg tgatcaaaaa ggagaactat tatgaagtaa
     1501 actcaaagtg ctatgaggag gcgaagaagc tgaagaagat ctccaatcag gccgagttca
     1561 tcgcttcctt ctataagaac gatctcatca agatcaatgg agagctttat cgcgtcattg
     1621 gtgtgaacaa tgacttgctg aacaggatcg aagtcaatat gatagacatt acctaccggg
     1681 agtatctcga aaacatgaat gataaacggc cgcctcacat catcaagaca atcgcatcta
     1741 aaactcagtc aataaaaaag tactctaccg atatcctggg gaatctctat gaagtgaagt
     1801 caaagaagca cccacaaatc attaaaaaag gtggatcccc caagaagaag aggaaagtct
     1861 cgagcgacta caaagaccat gacggtgatt ataaagatca tgacatcgat tacaaggatg
     1921 acgatgacaa gtctggtggt tctactaatc tgtcagatat tattgaaaag gagaccggta
     1981 agcaactggt tatccaggaa tccatcctca tgctcccaga ggaggtggaa gaagtcattg
     2041 ggaacaagcc ggaaagcgat atactcgtgc acaccgccta cgacgagagc accgacgaga
     2101 atgtcatgct tctgactagc gacgcccctg aatacaagcc ttgggctctg gtcatacagg
     2161 atagcaacgg tgagaacaag attaagatgc tctctggtgg ttctcccaag aagaagagga
     2221 aagtcggaag cggcgcaaca aacttctctc tgctgaaaca agccggagat gtcgaagaga
     2281 atcctggacc gatggtgtct aagggcgaag agctgattaa ggagaacatg cacatgaagc
     2341 tgtacatgga gggcaccgtg aacaaccacc acttcaagtg cacatccgag ggcgaaggca
     2401 agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct
     2461 tcgccttcga catcctggct accagcttca tgtacggcag cagaaccttc atcaaccaca
     2521 cccagggcat ccccgacttc tttaagcagt ccttccctga gggcttcaca tgggagagag
     2581 tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
     2641 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cccatccaac ggccctgtga
     2701 tgcagaagaa aacactcggc tgggaggcca acaccgagat gctgtacccc gctgacggcg
     2761 gcctggaagg cagaagcgac atggccctga agctcgtggg cgggggccac ctgatctgca
     2821 acttcaagac cacatacaga tccaagaaac ccgctaagaa cctcaagatg cccggcgtct
     2881 actatgtgga ccacagactg gaaagaatca aggaggccga caaagagacc tacgtcgagc
     2941 agcacgaggt ggctgtggcc agatactgcg acctccctag caaactgggg cacaaactta
     3001 attaactaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3061 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3121 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3181 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggaggt
     3241 accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     3301 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     3361 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     3421 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     3481 ggacgaaaca ccgccttccg agtctcccac tgcagtttta gtactctgga aacagaatct
     3541 actaaaacaa ggcaaaatgc cgtgtttatc tcgtcaactt gttggcgaga tttttgcggc
     3601 cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     3661 aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg
     3721 agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt acgcatctgt
     3781 gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt
     3841 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccttagc
     3901 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3961 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     4021 caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     4081 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     4141 aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggt
     4201 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     4261 aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     4321 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4381 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     4441 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     4501 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     4561 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4621 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4681 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4741 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4801 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4861 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     4921 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     4981 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5041 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     5101 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     5161 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     5221 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     5281 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     5341 gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca ttgcagcact
     5401 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5461 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     5521 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     5581 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     5641 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     5701 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     5761 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     5821 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     5881 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     5941 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6001 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6061 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     6121 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     6181 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     6241 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     6301 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     6361 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     6421 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     6481 gcctctcccc gcgcgttggc cgattcatta atgcag
//
