LOCUS       sequence-375885-        8960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SaAID2S 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_SaAID2S
FEATURES             Location/Qualifiers
     source          1..8960
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(90..678)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(170..189)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(849..1640,1641..1709))
                     /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     1472..1491
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1710..1814)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1977..1998)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2209..3387
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2440..3378
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3335..3355
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3408..3417
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3414..4349
                     /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    4379..4603
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4592..4609
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     regulatory      4702..4711
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4714..4734
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8320..8340
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8347..8601
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT
                     AYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     polyA_signal    8602..8650
                     /note="synthetic polyadenylation signal"
     primer_bind     8791..8810
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        join(8900..8960,1..15)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
ORIGIN
        1 aacttgttgg cgagattttt ttacgcgttg acattgatta ttgaccagca aaaggccagg
       61 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
      121 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
      181 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
      241 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
      301 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
      361 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
      421 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
      481 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
      541 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
      601 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
      661 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
      721 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
      781 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
      841 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
      901 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
      961 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     1021 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     1081 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     1141 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     1201 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     1261 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     1321 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     1381 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     1441 cctagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     1501 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     1561 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     1621 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     1681 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     1741 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     1801 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     1861 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     1921 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     1981 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     2041 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     2101 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2161 gaaagccgca gctaatggac cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc
     2221 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     2281 aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     2341 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     2401 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc
     2461 cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg
     2521 ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg
     2581 ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg
     2641 ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct
     2701 ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt
     2761 gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac
     2821 ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg
     2881 agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg
     2941 ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg
     3001 gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg
     3061 ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc
     3121 gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc
     3181 ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac
     3241 actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa
     3301 tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt
     3361 ttttttcttc catttcaggt gtcgtgagga atttcgaccc ggatccggcc accatggcgc
     3421 gtaaggtcga tctcacctcc tgcgatcgcg agccgatcca catccccggc agcattcagc
     3481 cgtgcggctg cctgctagcc tgcgacgcgc aggcggtgcg gatcacgcgc attacggaaa
     3541 atgccggcgc gttctttgga cgcgaaactc cgcgggtcgg tgagctactc gccgattact
     3601 tcggcgagac cgaagcccat gcgctgcgca acgcactggc gcagtcctcc gatccaaagc
     3661 gaccggcgct gatcttcggt tggcgcgacg gcctgaccgg ccgcaccttc gacatctcac
     3721 tgcatcgcca tgacggtaca tcgatcatcg agttcgagcc tgcggcggcc gaacaggccg
     3781 acaatccgct gcggctgacg cggcagatca tcgcgcgcac caaagaactg aagtcgctcg
     3841 aagagatggc cgcacgggtg ccgcgctatc tgcaggcgat gctcggctat caccgcgtga
     3901 tgttgtaccg cttcgcggac gacggctccg ggatggtgat cggcgaggcg aagcgcagcg
     3961 acctcgagag ctttctcggt cagcactttc cggcgtcgct ggtcccgcag caggcgcggc
     4021 tactgtactt gaagaacgcg atccgcgtgg tctcggattc gcgcggcatc agcagccgga
     4081 tcgtgcccga gcacgacgcc tccggcgccg cgctcgatct gtcgttcgcg cacctgcgca
     4141 gcatctcgcc ctgccatctc gaatttctgc ggaacatggg cgtcagcgcc tcgatgtcgc
     4201 tgtcgatcat cattgacggc acgctatggg gattgatcat ctgtcatcat tacgagccgc
     4261 gtgccgtgcc gatggcgcag cgcgtcgcgg ccgaaatgtt cgccgacttc ttatcgctgc
     4321 acttcaccgc cgcccaccac caacgctaag cggccgcgac tctaaccggt ctcagaagcc
     4381 atagagccca ccgcatcccc agcatgcctg ctattgtctt cccaatcctc ccccttgctg
     4441 tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt
     4501 tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg
     4561 ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatgtactt
     4621 atataagggg gtgggggcgc gttcgtcctc agtcgcgatc gaacactcga gccgagcaga
     4681 cgtgcctacg gaccgtctag agccaccatg gcaccgaaga agaagcgtaa agtcggaatc
     4741 cacggagttc ctgcggcatc acaccgctgc tacgtgctgt ttgagttgaa gcgaaggggc
     4801 gaaagaaggg cttgcttttg gggctatgcc gtcaacaagc cccaaagtgg caccgagaga
     4861 ggaatacacg ctgagatatt cagtatccga aaggtggaag agtatcttcg ggataatcct
     4921 gggcagttta cgatcaactg gtattccagc tggagtcctt gcgctgattg tgccgagaaa
     4981 attctggaat ggtataatca ggaacttcgg ggaaacgggc acacattgaa aatcgaagcc
     5041 tgcaagctgt actacgagaa gaatgcccgg aaccagatag gactccagaa tctgagggac
     5101 aatggtgtag gcctgaacgt gggtggaggt ggcagtgatg tcggcggagg ctccaagcga
     5161 aattacattc tggggctggc cattggcatt acatcagtgg gctatggcat cattgactac
     5221 gagacaaggg acgtgatcga cgccggcgtg agactgttca aggaggccaa cgtggagaac
     5281 aatgagggcc ggagatccaa gaggggagca aggcgcctga agcggagaag gcgccacaga
     5341 atccagagag tgaagaagct gctgttcgat tacaacctgc tgaccgacca ctccgagctg
     5401 tctggcatca atccttatga ggccagagtg aagggcctgt cccagaagct gtctgaggag
     5461 gagtttagcg ccgccctgct gcacctggca aagaggagag gcgtgcacaa cgtgaatgag
     5521 gtggaggagg acaccggcaa cgagctgtcc acaaaggagc agatcagccg caattccaag
     5581 gccctggagg agaagtatgt ggccgagctg cagctggagc ggctgaagaa ggatggcgag
     5641 gtgaggggct ccatcaatcg cttcaagacc tctgactacg tgaaggaggc caagcagctg
     5701 ctgaaggtgc agaaggccta ccaccagctg gatcagtcct ttatcgatac atatatcgac
     5761 ctgctggaga caaggcgcac atactatgag ggaccaggag agggctctcc cttcggctgg
     5821 aaggacatca aggagtggta cgagatgctg atgggccact gcacctattt tccagaggag
     5881 ctgagaagcg tgaagtacgc ctataacgcc gatctgtaca acgccctgaa tgacctgaac
     5941 aacctggtca tcaccaggga tgagaacgag aagctggagt actatgagaa gttccagatc
     6001 atcgagaacg tgttcaagca gaagaagaag cctacactga agcagatcgc caaggagatc
     6061 ctggtgaacg aggaggacat caagggctac cgcgtgacct ccacaggcaa gccagagttc
     6121 accaatctga aggtgtatca cgatatcaag gacatcacag cccggaagga gatcatcgag
     6181 aacgccgagc tgctggatca gatcgccaag atcctgacca tctatcagag ctccgaggac
     6241 atccaggagg agctgaccaa cctgaatagc gagctgacac aggaggagat cgagcagatc
     6301 agcaatctga agggctacac cggcacacac aacctgagcc tgaaggccat caatctgatc
     6361 ctggatgagc tgtggcacac aaacgacaat cagatcgcca tctttaaccg gctgaagctg
     6421 gtgccaaaga aggtggacct gtcccagcag aaggagatcc caaccacact ggtggacgat
     6481 ttcatcctgt ctcccgtggt gaagcggagc ttcatccaga gcatcaaagt gatcaacgcc
     6541 atcatcaaga agtacggcct gcccaatgat atcatcatcg agctggccag ggagaagaac
     6601 tccaaggacg cccagaagat gatcaatgag atgcagaaga ggaaccgcca gaccaatgag
     6661 cggatcgagg agatcatcag aaccacaggc aaggagaacg ccaagtacct gatcgagaag
     6721 atcaagctgc acgatatgca ggagggcaag tgtctgtatt ctctggaggc catccctctg
     6781 gaggacctgc tgaacaatcc attcaactac gaggtggatc acatcatccc ccggagcgtg
     6841 agcttcgaca attcttttaa caataaggtg ctggtgaagc aggaggagaa cagcaagaag
     6901 ggcaatagga cccctttcca gtacctgtct agctccgatt ctaagatcag ctacgagaca
     6961 ttcaagaagc acatcctgaa tctggccaag ggcaagggcc gcatcagcaa gaccaagaag
     7021 gagtacctgc tggaggagcg ggacatcaac agattctccg tgcagaagga cttcatcaac
     7081 cggaatctgg tggacaccag atacgccaca cgcggcctga tgaatctgct gcggtcttat
     7141 ttcagagtga acaatctgga tgtgaaggtg aagagtatca acggcggctt cacctccttt
     7201 ctgcggagaa agtggaagtt taagaaggag cgcaacaagg gctataagca ccacgccgag
     7261 gatgccctga tcatcgccaa tgccgacttc atctttaagg agtggaagaa gctggacaag
     7321 gccaagaaag tgatggagaa ccagatgttc gaggagaagc aggccgagag catgcccgag
     7381 atcgagacag agcaggagta caaggagatt ttcatcacac ctcaccagat caagcacatc
     7441 aaggacttca aggactacaa gtattctcac agggtggata agaagcccaa ccgcgagctg
     7501 atcaatgaca ccctgtatag cacacggaag gacgataagg gcaataccct gatcgtgaac
     7561 aatctgaacg gcctgtacga caaggataat gacaagctga agaagctgat caacaagtct
     7621 cccgagaagc tgctgatgta ccaccacgat cctcagacat atcagaagct gaagctgatc
     7681 atggagcagt acggcgacga gaagaaccca ctgtataagt actatgagga gacaggcaac
     7741 tacctgacaa agtatagcaa gaaggataat ggccccgtga tcaagaagat caagtactat
     7801 ggcaacaagc tgaatgccca cctggacatc accgacgatt accctaactc tcgcaataag
     7861 gtggtgaagc tgagcctgaa gccataccgg ttcgacgtgt acctggacaa cggcgtgtat
     7921 aagtttgtga cagtgaagaa tctggatgtg atcaagaagg agaactacta tgaggtgaac
     7981 agcaagtgct acgaggaggc caagaagctg aagaagatca gcaaccaggc cgagttcatc
     8041 gcctcttttt acaacaatga cctgatcaag atcaatggcg agctgtatag agtgatcggc
     8101 gtgaacaatg atctgctgaa cagaatcgaa gtgaatatga tcgacatcac ctacagggag
     8161 tatctggaga acatgaatga taagaggccc cctcgcatca tcaagaccat cgcctctaag
     8221 acacagagca tcaagaagta cagcacagac atcctgggga acctgtatga agtcaagagc
     8281 aagaaacatc ctcagattat caagaaaggc ggtggaggtc ccaagaagaa aagaaaagtc
     8341 ggtaccatga ccaacctttc cgacatcata gagaaggaaa caggcaaaca gttggtcatc
     8401 caagagtcga tactcatgct tcctgaagaa gttgaggagg tcattgggaa taagccggaa
     8461 agtgacattc tcgtacacac tgcgtatgat gagagcaccg atgagaacgt gatgctgctc
     8521 acgtcagatg ccccagagta caaaccctgg gctctggtga ttcaggactc taatggagag
     8581 aacaagatca agatgctata gaataaaaga tctttatttt cattagatct gtgtgttggt
     8641 tttttgtgtg aattcgaacg ctgacgtcat caacccgctc caaggaatcg cgggcccagt
     8701 gtcactaggc gggaacaccc agcgcgcgtg cgccctggca ggaagatggc tgtgagggac
     8761 aggggagtgg cgccctgcaa tatttgcatg tcgctatgtg ttctgggaaa tcaccataaa
     8821 cgtgaaatgt ctttggattt gggaatctta taagttctgt atgaggacca cagatccccg
     8881 atgttccaat cagtacgcag ttttagtact ctggaaacag aatctactaa aacaaggcaa
     8941 aatgccgtgt ttatctcgtc
//
