enIscB

LOCUS      enIscB       7501 bp DNA     circular SYN 25-AUG-2024
DEFINITION  Vector encoding human codon-optimized enhanced-activity OgeuIscB 
            driven by CAG promoter, optimized _RNA (_RNA*) driven by hU6, and 
            mCherry driven by CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7501)
  AUTHORS   Han D, Xiao Q, Wang Y, Zhang H, Dong X, Li G, Kong X, Wang S, Song 
            J, Zhang W, Zhou J, Bi L, Yuan Y, Shi L, Zhong N, Yang H, Zhou Y
  TITLE     Development of miniature base editors using engineered IscB nickase.
  JOURNAL   Nat Methods. 2023 May 25. doi: 10.1038/s41592-023-01898-9.
  PUBMED    37231266
REFERENCE   2  (bases 1 to 7501)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2024 from SnapGene Server 7.0.3
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2023 May 25. doi: 10.1038/s41592-023-01898-9."
FEATURES             Location/Qualifiers
     source          1..7501
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     108..127
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        227..512
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        514..791
                     /label=chicken beta-actin promoter
     intron          792..1019
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1031..1040
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1040..1105
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1109..1129
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2618..2665
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(2693..2710)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2699..2906
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2907..3155
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2907..2927
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3078..3097
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        3446..3749
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3750..3953
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3904..3924
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3982..4692
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4127..4145)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     rep_origin      5247..5702
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5334..5353)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5544..5565
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5719..5738)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5838..5860
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5898..5916)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5984..6088
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6089..6949
                     /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(6307..6326)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7120..7501,1..207)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
       61 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
      121 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
      181 tcgtcagggg ggcggagcct atggaaaaac gccagcaaca ggtacccgtt acataactta
      241 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaatagtaa
      301 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      361 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      421 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      481 acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg ttctgcttca
      541 ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat
      601 tttgtgcagc gatgggggcg gggggggggg gggggcgcgc gccaggcggg gcggggcggg
      661 gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc
      721 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg
      781 cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct
      841 cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg
      901 gcccttctcc tccgggctgt aattagctga gcaagaggta agggtttaag ggatggttgg
      961 ttggtggggt attaatgttt aattacctgg agcacctgcc tgaaatcact ttttttcagg
     1021 ttggaccggt gccaccatgg actataagga ccacgacgga gactacaagg atcatgatat
     1081 tgattacaaa gacgatgacg ataagatgcc taagaagaag agaaaggtga tgatggccgt
     1141 ggtgtacgtg atcagcaagt ctggcaaacc cctgatgccc accacccggt gcggccacgt
     1201 gagaatcctg ctgaaggagg gcaaggccag agtggtggag cggaaaccgt tcaccatcca
     1261 gctgacctac gagagcgcgg aggaaacaca gcctctggtc ctgggcattg atcctggacg
     1321 caccaatatc ggcatgagcg tggtgacaga atccggagag agcgtgttca acgcccagat
     1381 cagaaccaga aacaaggacg tgcctaagct gatgaaagac agaaagcagt accggatggc
     1441 tcacagacgg ctgaagcggc gctgcaagcg aagaagaaga gccaaggccg ccggcaccgc
     1501 attcgaggaa ggagaaaagc agagactgct gcctggctgt ttcaagccta tcacatgtaa
     1561 gagcatcaga aataaggaag ctagattcaa taacaggaag cggcctgtgg gatggctgac
     1621 cccaaccgcc aaccacctgc tggtgaccca cctgaacgtg gtcaagaagg tgcaaaagat
     1681 cctgcctgtg gccaaggtgg tgctggaact gaatcggttc agcttcatgg ccatgaacaa
     1741 ccccaaggtg cagcggtggc agtaccagag aggccctctg tacggtaaag gcagcgtgga
     1801 agaggccgtg agcatgcagc aggacggcca ctgcctgttt tgcaagcacg gcattgacca
     1861 ctatcaccat gtggttccta ggagaaagaa cggctctgaa accctggaga accgggtggg
     1921 cctgtgcgag gaacaccacc ggctcgtcca cacagataag gagtgggagg ccaatctggc
     1981 ttctaagaag agcggaatga acaagaaata ccacgccctg agcgtgctga accagatcat
     2041 cccatacctg gccgaccagc ttgccgatat gttccccggc aacttctgtg tgaccagcgg
     2101 acaggataca tacctgtttc gggaagaaca tggcatcccc aaggaccact acctggatgc
     2161 ctactgcatc gcctgcagcg ccctgaccga cgccaagaag gtgagcagcc ctaaaggcag
     2221 accttacatg gtgagacagt tcagacgcca cgatagacag gcctgtcaca aggccaacct
     2281 gaacagaaga tactacatgg gcggcaaact ggttgctaca aacagacata aggccatgga
     2341 ccagaaaacc gacagcctgg aggagtacag agccgcccac agcgccgctg acgtatccaa
     2401 gctgacagtg aagcaccctt ctgctcaata taaggacatg tcccggatca tgcctggcag
     2461 catcctcgtg tccggcgagg gcaagctctt tacactgaga agatccgagg gcagaaacaa
     2521 gggccaagtg aactacttcg tgagcaccga gggcatcaag tactgggcca gaaagtgcca
     2581 gtatctgaga aacaatggcg gcctgcagat ctacgtgaaa aggccggcgg ccacgaagaa
     2641 ggccggccag gcaaagaaga aaaagtaaga attcctagag ctcgctgatc agcctcgact
     2701 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2761 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     2821 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2881 gaagagaata gcaggcatgc tggggagagg gcctatttcc catgattcct tcatatttgc
     2941 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     3001 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     3061 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     3121 tggctttata tatcttgtgg aaaggacgaa acaccgggag tcttccgaag acttggctcg
     3181 tccaactgcg gttgaacgag cacaggctga gacattcgta aggccgaaag gccggacgca
     3241 ccctgggatt tccccagtcc ccggaactgc atagcggatg ccagttgatg gagcaatcta
     3301 tcagataagc cagggggaac aatcacctct ctgtatcaga gagagtttta caaaaggagg
     3361 aacggtttta tttttttgcg gccgctagtt attaatagta atcaattacg gggtcattag
     3421 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     3481 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     3541 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     3601 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     3661 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     3721 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     3781 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     3841 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     3901 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
     3961 tgaaccgtca gatccgctag catggtgagc aagggcgagg aggataacat ggccatcatc
     4021 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     4081 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     4141 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
     4201 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
     4261 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     4321 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
     4381 ttcccctccg acggccccgt aatgcagaag aaaaccatgg gctgggaggc ctcctccgag
     4441 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
     4501 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag
     4561 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac
     4621 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag
     4681 ctgtacaagt aaggatccgg gatgcagaaa ttgatgatct attaaacaat aaagatgtcc
     4741 actaaaatgg aagtttttcc tgtcatactt tgttaagaag ggtgagaaca gagtacctac
     4801 attttgaatg gaaggattgg agctacgggg gtgggggtgg ggtgggatta gataaatgcc
     4861 tgctctttac tgaaggctct ttactattgc tttatgataa tgtttcatag ttggatatca
     4921 taatttaaac aagcaaaacc aaattaaggg ccagctcatt cctcccactc atgatctata
     4981 gatctataga tctctcgtgg gatcattgtt tttctcttga ttcccacttt gtggttctaa
     5041 gtactgtggt ttccaaatgt gtcagtttca tagcctgaag aacgagatca gcagcctctg
     5101 ttccacatac acttcattct cagtattgtt ttgccaagtt ctaattccat cagaagctgg
     5161 tcgacctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     5221 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     5281 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     5341 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     5401 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     5461 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5521 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     5581 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     5641 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     5701 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     5761 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5821 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5881 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     5941 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     6001 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     6061 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     6121 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     6181 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     6241 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     6301 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     6361 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     6421 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6481 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6541 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6601 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6661 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6721 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6781 taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg ggccagatgg
     6841 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6901 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6961 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     7021 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     7081 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     7141 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     7201 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     7261 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     7321 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     7381 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     7441 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7501 a
//