LOCUS       sequence-375883-        8960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SaAID3S and gRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8960)
  AUTHORS   Li A, Mitsunobu H, Yoshioka S, Suzuki T, Kondo A, Nishida K
  TITLE     Cytosine base editing systems with minimized off-target effect and 
            molecular size.
  JOURNAL   Nat Commun. 2022 Aug 8;13(1):4531. doi: 10.1038/s41467-022-32157-8.
  PUBMED    35941130
REFERENCE   2  (bases 1 to 8960)
  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 
            Commun."; date: "2022-08-8"; volume: "13(1):4531. doi"; pages: " 
            10.1038/s41467-022-32157-8"
COMMENT     Sequence Label: Mammalian_SaAID3S
FEATURES             Location/Qualifiers
     source          1..8960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(11..599)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(91..110)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(770..1561,1562..1630))
                     /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     1393..1412
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1631..1735)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1898..1919)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2130..3308
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2361..3299
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3256..3276
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3329..3338
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3335..4270
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="MARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAG
                     AFFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLH
                     RHDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVM
                     LYRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSR
                     IVPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYE
                     PRAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     polyA_signal    4300..4524
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4513..4530
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     regulatory      4623..4632
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4635..4655
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8241..8261
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8268..8522
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
                     AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     polyA_signal    8523..8571
                     /note="synthetic polyadenylation signal"
     primer_bind     8712..8731
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        8821..8896
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
ORIGIN
        1 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
       61 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
      121 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
      181 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
      241 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
      301 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
      361 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
      421 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
      481 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
      541 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
      601 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
      661 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
      721 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
      781 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
      841 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
      901 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
      961 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     1021 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     1081 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     1141 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     1201 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     1261 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     1321 agtactcaac caagtcattc tgagaatagt gtatgcggcg acctagttgc tcttgcccgg
     1381 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     1441 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     1501 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     1561 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     1621 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     1681 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     1741 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     1801 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     1861 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     1921 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     1981 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     2041 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgc agctaatgga
     2101 ccttctaggt cttgaaagga gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca
     2161 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
     2221 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     2281 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     2341 ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta
     2401 cgggttatgg cccttgcgtg ccttgaatta cttccacctg gctgcagtac gtgattcttg
     2461 atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc
     2521 cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct
     2581 ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt
     2641 gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc
     2701 tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc
     2761 gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt
     2821 ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct
     2881 gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg
     2941 gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt
     3001 cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg
     3061 agtaccgggc gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt
     3121 taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg
     3181 aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg
     3241 gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg
     3301 tgtcgtgagg aatttcgacc cggatccggc caccatggcg cgtaaggtcg atctcacctc
     3361 ctgcgatcgc gagccgatcc acatccccgg cagcattcag ccgtgcggct gcctgctagc
     3421 ctgcgacgcg caggcggtgc ggatcacgcg cattacggaa aatgccggcg cgttctttgg
     3481 acgcgaaact ccgcgggtcg gtgagctact cgccgattac ttcggcgaga ccgaagccca
     3541 tgcgctgcgc aacgcactgg cgcagtcctc cgatccaaag cgaccggcgc tgatcttcgg
     3601 ttggcgcgac ggcctgaccg gccgcacctt cgacatctca ctgcatcgcc atgacggtac
     3661 atcgatcatc gagttcgagc ctgcggcggc cgaacaggcc gacaatccgc tgcggctgac
     3721 gcggcagatc atcgcgcgca ccaaagaact gaagtcgctc gaagagatgg ccgcacgggt
     3781 gccgcgctat ctgcaggcga tgctcggcta tcaccgcgtg atgttgtacc gcttcgcgga
     3841 cgacggctcc gggatggtga tcggcgaggc gaagcgcagc gacctcgaga gctttctcgg
     3901 tcagcacttt ccggcgtcgc tggtcccgca gcaggcgcgg ctactgtact tgaagaacgc
     3961 gatccgcgtg gtctcggatt cgcgcggcat cagcagccgg atcgtgcccg agcacgacgc
     4021 ctccggcgcc gcgctcgatc tgtcgttcgc gcacctgcgc agcatctcgc cctgccatct
     4081 cgaatttctg cggaacatgg gcgtcagcgc ctcgatgtcg ctgtcgatca tcattgacgg
     4141 cacgctatgg ggattgatca tctgtcatca ttacgagccg cgtgccgtgc cgatggcgca
     4201 gcgcgtcgcg gccgaaatgt tcgccgactt cttatcgctg cacttcaccg ccgcccacca
     4261 ccaacgctaa gcggccgcga ctctaaccgg tctcagaagc catagagccc accgcatccc
     4321 cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc
     4381 ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa
     4441 ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag
     4501 atggctggca actagaaggc acagtcgagg ctgatgtact tatataaggg ggtgggggcg
     4561 cgttcgtcct cagtcgcgat cgaacactcg agccgagcag acgtgcctac ggaccgtcta
     4621 gagccaccat ggcaccgaag aagaagcgta aagtcggaat ccacggagtt cctgcggcaa
     4681 agcgaaatta cattctgggg ctggccattg gcattacatc agtgggctat ggcatcattg
     4741 actacgagac aagggacgtg atcgacgccg gcgtgagact gttcaaggag gccaacgtgg
     4801 agaacaatga gggccggaga tccaagaggg gagcaaggcg cctgaagcgg agaaggcgcc
     4861 acagaatcca gagagtgaag aagctgctgt tcgattacaa cctgctgacc gaccactccg
     4921 agctgtctgg catcaatcct tatgaggcca gagtgaaggg cctgtcccag aagctgtctg
     4981 aggaggagtt tagcgccgcc ctgctgcacc tggcaaagag gagaggcgtg cacaacgtga
     5041 atgaggtgga ggaggacacc ggcaacgagc tgtccacaaa ggagcagatc agccgcaatt
     5101 ccaaggccct ggaggagaag tatgtggccg agctgcagct ggagcggctg aagaaggatg
     5161 gcgaggtgag gggctccatc aatcgcttca agacctctga ctacgtgaag gaggccaagc
     5221 agctgctgaa ggtgcagaag gcctaccacc agctggatca gtcctttatc gatacatata
     5281 tcgacctgct ggagacaagg cgcacatact atgagggacc aggagagggc tctcccttcg
     5341 gctggaagga catcaaggag tggtacgaga tgctgatggg ccactgcacc tattttccag
     5401 aggagctgag aagcgtgaag tacgcctata acgccgatct gtacaacgcc ctgaatgacc
     5461 tgaacaacct ggtcatcacc agggatgaga acgagaagct ggagtactat gagaagttcc
     5521 agatcatcga gaacgtgttc aagcagaaga agaagcctac actgaagcag atcgccaagg
     5581 agatcctggt gaacgaggag gacatcaagg gctaccgcgt gacctccaca ggcaagccag
     5641 agttcaccaa tctgaaggtg tatcacgata tcaaggacat cacagcccgg aaggagatca
     5701 tcgagaacgc cgagctgctg gatcagatcg ccaagatcct gaccatctat cagagctccg
     5761 aggacatcca ggaggagctg accaacctga atagcgagct gacacaggag gagatcgagc
     5821 agatcagcaa tctgaagggc tacaccggca cacacaacct gagcctgaag gccatcaatc
     5881 tgatcctgga tgagctgtgg cacacaaacg acaatcagat cgccatcttt aaccggctga
     5941 agctggtgcc aaagaaggtg gacctgtccc agcagaagga gatcccaacc acactggtgg
     6001 acgatttcat cctgtctccc gtggtgaagc ggagcttcat ccagagcatc aaagtgatca
     6061 acgccatcat caagaagtac ggcctgccca atgatatcat catcgagctg gccagggaga
     6121 agaactccaa ggacgcccag aagatgatca atgagatgca gaagaggaac cgccagacca
     6181 atgagcggat cgaggagatc atcagaacca caggcaagga gaacgccaag tacctgatcg
     6241 agaagatcaa gctgcacgat atgcaggagg gcaagtgtct gtattctctg gaggccatcc
     6301 ctctggagga cctgctgaac aatccattca actacgaggt ggatcacatc atcccccgga
     6361 gcgtgagctt cgacaattct tttaacaata aggtgctggt gaagcaggag gagaacagca
     6421 agaagggcaa taggacccct ttccagtacc tgtctagctc cgattctaag atcagctacg
     6481 agacattcaa gaagcacatc ctgaatctgg ccaagggcaa gtcacaccgc tgctacgtgc
     6541 tgtttgagtt gaagcgaagg ggcgaaagaa gggcttgctt ttggggctat gccgtcaaca
     6601 agccccaaag tggcaccgag agaggaatac acgctgagat attcagtatc cgaaaggtgg
     6661 aagagtatct tcgggataat cctgggcagt ttacgatcaa ctggtattcc agctggagtc
     6721 cttgcgctga ttgtgccgag aaaattctgg aatggtataa tcaggaactt cggggaaacg
     6781 ggcacacatt gaaaatcgaa gcctgcaagc tgtactacga gaagaatgcc cggaaccaga
     6841 taggactcca gaatctgagg gacaatggtg taggcctgaa cgtgggtgga ggtggcagtg
     6901 atgtcggcgg aggctccggc cgcatcagca agaccaagaa ggagtacctg ctggaggagc
     6961 gggacatcaa cagattctcc gtgcagaagg acttcatcaa ccggaatctg gtggacacca
     7021 gatacgccac acgcggcctg atgaatctgc tgcggtctta tttcagagtg aacaatctgg
     7081 atgtgaaggt gaagagtatc aacggcggct tcacctcctt tctgcggaga aagtggaagt
     7141 ttaagaagga gcgcaacaag ggctataagc accacgccga ggatgccctg atcatcgcca
     7201 atgccgactt catctttaag gagtggaaga agctggacaa ggccaagaaa gtgatggaga
     7261 accagatgtt cgaggagaag caggccgaga gcatgcccga gatcgagaca gagcaggagt
     7321 acaaggagat tttcatcaca cctcaccaga tcaagcacat caaggacttc aaggactaca
     7381 agtattctca cagggtggat aagaagccca accgcgagct gatcaatgac accctgtata
     7441 gcacacggaa ggacgataag ggcaataccc tgatcgtgaa caatctgaac ggcctgtacg
     7501 acaaggataa tgacaagctg aagaagctga tcaacaagtc tcccgagaag ctgctgatgt
     7561 accaccacga tcctcagaca tatcagaagc tgaagctgat catggagcag tacggcgacg
     7621 agaagaaccc actgtataag tactatgagg agacaggcaa ctacctgaca aagtatagca
     7681 agaaggataa tggccccgtg atcaagaaga tcaagtacta tggcaacaag ctgaatgccc
     7741 acctggacat caccgacgat taccctaact ctcgcaataa ggtggtgaag ctgagcctga
     7801 agccataccg gttcgacgtg tacctggaca acggcgtgta taagtttgtg acagtgaaga
     7861 atctggatgt gatcaagaag gagaactact atgaggtgaa cagcaagtgc tacgaggagg
     7921 ccaagaagct gaagaagatc agcaaccagg ccgagttcat cgcctctttt tacaacaatg
     7981 acctgatcaa gatcaatggc gagctgtata gagtgatcgg cgtgaacaat gatctgctga
     8041 acagaatcga agtgaatatg atcgacatca cctacaggga gtatctggag aacatgaatg
     8101 ataagaggcc ccctcgcatc atcaagacca tcgcctctaa gacacagagc atcaagaagt
     8161 acagcacaga catcctgggg aacctgtatg aagtcaagag caagaaacat cctcagatta
     8221 tcaagaaagg cggtggaggt cccaagaaga aaagaaaagt cggtaccatg accaaccttt
     8281 ccgacatcat agagaaggaa acaggcaaac agttggtcat ccaagagtcg atactcatgc
     8341 ttcctgaaga agttgaggag gtcattggga ataagccgga aagtgacatt ctcgtacaca
     8401 ctgcgtatga tgagagcacc gatgagaacg tgatgctgct cacgtcagat gccccagagt
     8461 acaaaccctg ggctctggtg attcaggact ctaatggaga gaacaagatc aagatgctat
     8521 agaataaaag atctttattt tcattagatc tgtgtgttgg ttttttgtgt gaattcgaac
     8581 gctgacgtca tcaacccgct ccaaggaatc gcgggcccag tgtcactagg cgggaacacc
     8641 cagcgcgcgt gcgccctggc aggaagatgg ctgtgaggga caggggagtg gcgccctgca
     8701 atatttgcat gtcgctatgt gttctgggaa atcaccataa acgtgaaatg tctttggatt
     8761 tgggaatctt ataagttctg tatgaggacc acagatcccc gatgttccaa tcagtacgca
     8821 gttttagtac tctggaaaca gaatctacta aaacaaggca aaatgccgtg tttatctcgt
     8881 caacttgttg gcgagatttt tttacgcgtt gacattgatt attgaccagc aaaaggccag
     8941 gaaccgtaaa aaggccgcgt
//
