LOCUS       sequence-496501-        8552 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  Vector encoding human codon-optimized engineered DelIscB nickase 
            fusion with TadA8e driven by CAG promoter, optimized sgRNA 
            (sgRNA-V5) 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 8552)
  TITLE     Engineering a CRISPR-associated IscB system for developing miniature
            genome-editing tools in human cells and mouse embryos
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8552)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 7, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: IABE
FEATURES             Location/Qualifiers
     source          1..8552
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      30..618
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     519..538
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        638..923
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        925..1202
                     /label=chicken beta-actin promoter
     intron          1203..1430
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3470..3490
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4046..4066
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4094..4111)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4100..4307
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4308..4556
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4308..4328
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4479..4498
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        4908..5211
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5212..5415
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5366..5386
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             5444..6154
                     /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(5589..5607)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     rep_origin      6709..7164
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6796..6815)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7006..7027
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7181..7200)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7300..7322
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7360..7378)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7446..7550
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7551..7619,7620..8411)
                     /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(7769..7788)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
       61 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
      121 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
      181 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
      241 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
      301 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
      361 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
      421 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
      481 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
      541 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
      601 gggcggagcc tatggaaaaa cgccagcaac aggtacccgt tacataactt acggtaaatg
      661 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag
      721 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      781 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      841 cctggcattg tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      901 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      961 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
     1021 cgatgggggc gggggggggg ggggggcgcg cgccaggsgg ggsggggsgg ggsgaggggs
     1081 ggggsggggs gaggsggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
     1141 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
     1201 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
     1261 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
     1321 ctccgggctg taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg
     1381 tattaatgtt taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg
     1441 tgccaccatg agcagagtgc tggtggtgga tgctgatagg cgccctctgg ccccttgcac
     1501 ccctcggaga gccagactgc tgctgaattc tggcaaagct gccgtgctga gacggtaccc
     1561 tttcaccatc atcctgaagc agagctaccc caccgccagc cctagacccg tgcggctgaa
     1621 gctggccccc ggaagcaaga ccaccggcat cgccgtggtg acagaggcca caggagaagt
     1681 ggtgtgggcc gctgagctgc agcacagagg ccagctgatc aagaacgccc tggaaaagag
     1741 aagagccctg cggcgcagcc ggcgaaaccg gaagacacgg tatagacctg ccagattcct
     1801 gaatagaaag agaacaggac ctccactgct gagcagcgcc gacactgtgt ctaccctggg
     1861 caagtggttg cccccgagcc tgcagcatag gatcgaggtg atcatgacct gggtccacag
     1921 actgagaaga tacctgccca tcaccgccat ttctcaggag atcgtgagat tcgacatgca
     1981 aaagatgcag aaccccgaaa tcagcggcgt ggaataccag cagggcaccc tcttcggcta
     2041 cgaggtgcgg gaatacctcc tggacaagtg gcggagacaa tgtggctact gcggcgctaa
     2101 ggacaagaga cttgaggtcg accacatcgt tcctcggagc cacggcggca gcgacagagt
     2161 gtccaacctg acactgtcct gcgagccttg caacaaaaga aagaaccaga ggccagctgc
     2221 tgtgttcctg gctaaaaagc ccgaggtcct gcagaagctg cagcggcagg ccaaggcccc
     2281 tctgaaggac gccgccgccg tgaacagcac ccgttacgcc ctgctggaaa gactgaaggc
     2341 caccggcctg cctgtggaag tggccagcgg cggaagaacc aagttcaata gaagcaggag
     2401 acagatcccc aagacccact ggctggatgc cgcctgcgtg ggcgcttcta cacctgaggt
     2461 gctgaagtgg gaggccgtta gaccactggc catcaaggcc atgggccacg gcaaacggca
     2521 ggtagtcaac gtggacgcct acggatttcc tcggggcaag gccaaaggca tccctgtgca
     2581 ccccttcaga accggagata tcgtgcgggc cgagattcca aagggcaaat acaagggaac
     2641 atacgtgtcc agaatcgccg agacaaccac atctaagcct ctagcaggct ttaagagcaa
     2701 gaccggcaag cggatccagt gtcacaccaa gcacatgaca aagctgttca accgcgacgg
     2761 ctatggctac ggcttcctga aagcccctga acctagacag accgtgatca gcgagtcttc
     2821 tggaggatct agcggaggct cctctggcag cgagacacca ggaacaagcg agtcagcaac
     2881 accagagagc agtggcggca gcagcggcgg cagctctgag gtggagtttt cccacgagta
     2941 ctggatgaga catgccctga ccctggccaa gagggcacgg gatgagaggg aggtgcctgt
     3001 gggagccgtg ctggtgctga acaatagagt gatcggcgag ggctggaaca gagccatcgg
     3061 cctacacgac ccaacagccc atgccgaaat tatggccctg agacaaggcg gcctggtcat
     3121 gcagaactac agactgattg acgccaccct gtacgtgaca ttcgagcctt gcgtgatgtg
     3181 cgctggcgcc atgatccact ctaggatcgg ccgcgtggtg tttggatgga gaaattctaa
     3241 aagaggcgcc gcaggctccc tgatgaacgt gctgaactac cccggcatga atcaccgcgt
     3301 cgaaattacc gagggaatcc tggcagatga atgtgccgcc ctgctgtgcg acttctatcg
     3361 gatgcctaga caggtgttca atgctcagaa gaaggcccag agttccatca actctggagg
     3421 atctagcgga ggatccaaga gaaccgctga tggctcagag tttgagagcc caaaaaagaa
     3481 aagaaaagtc agtggcggca gcagcggcgg cagctctgag gtggagtttt cccacgagta
     3541 ctggatgaga catgccctga ccctggccaa gagggcacgg gatgagaggg aggtgcctgt
     3601 gggagccgtg ctggtgctga acaatagagt gatcggcgag ggctggaaca gagccatcgg
     3661 cctacacgac ccaacagccc atgccgaaat tatggccctg agacaaggcg gcctggtcat
     3721 gcagaactac agactgattg acgccaccct gtacgtgaca ttcgagcctt gcgtgatgtg
     3781 cgctggcgcc atgatccact ctaggatcgg ccgcgtggtg tttggatgga gaaattctaa
     3841 aagaggcgcc gcaggctccc tgatgaacgt gctgaactac cccggcatga atcaccgcgt
     3901 cgaaattacc gagggaatcc tggcagatga atgtgccgcc ctgctgtgcg acttctatcg
     3961 gatgcctaga caggtgttca atgctcagaa gaaggcccag agttccatca acaagagaac
     4021 cgccgacggc tccgagttcg agagccccaa gaagaagcgc aaagtgtgag aattcctaga
     4081 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     4141 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     4201 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     4261 gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagag ggcctatttc
     4321 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     4381 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     4441 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     4501 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgggg
     4561 agaccgagag agggtctcag ttgtagcctc cactcattga aaaatgagtg cgggaaaccg
     4621 cagctacaac cagactaagt tctttgggaa ctacgttatt gtcgtcatca tacccacggg
     4681 tgcgtgccag cctgtggctc tatggtcgtg cattaaacag ggactttgga gggtgcccag
     4741 tgtgtacggc gtcaaaagcg gcgataacat tgtcgaggca aactttacgt gacggcgaaa
     4801 gcccgatcgc tagctttaag gctaattttt tatttttttg cggccgctag ttattaatag
     4861 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     4921 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     4981 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     5041 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     5101 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     5161 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     5221 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     5281 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     5341 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     5401 tatataagca gagctggttt agtgaaccgt cagatccgct agcatggtga gcaagggcga
     5461 ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc
     5521 cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     5581 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct
     5641 gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga
     5701 ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     5761 cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa
     5821 ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaaaaccat
     5881 gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat
     5941 caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta
     6001 caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat
     6061 cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca
     6121 ctccaccggc ggcatggacg agctgtacaa gtaaggatcc gggatgcaga aattgatgat
     6181 ctattaaaca ataaagatgt ccactaaaat ggaagttttt cctgtcatac tttgttaaga
     6241 agggtgagaa cagagtacct acattttgaa tggaaggatt ggagctacgg gggtgggggt
     6301 ggggtgggat tagataaatg cctgctcttt actgaaggct ctttactatt gctttatgat
     6361 aatgtttcat agttggatat cataatttaa acaagcaaaa ccaaattaag ggccagctca
     6421 ttcctcccac tcatgatcta tagatctata gatctctcgt gggatcattg tttttctctt
     6481 gattcccact ttgtggttct aagtactgtg gtttccaaat gtgtcagttt catagcctga
     6541 agaacgagat cagcagcctc tgttccacat acacttcatt ctcagtattg ttttgccaag
     6601 ttctaattcc atcagaagct ggtcgacctg caggggcgcc tgatgcggta ttttctcctt
     6661 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     6721 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     6781 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     6841 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     6901 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     6961 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     7021 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     7081 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     7141 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     7201 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     7261 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     7321 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     7381 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     7441 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     7501 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     7561 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     7621 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     7681 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     7741 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     7801 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     7861 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     7921 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     7981 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     8041 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     8101 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     8161 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     8221 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca
     8281 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     8341 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     8401 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     8461 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     8521 cttaacgtga gttttcgttc cactgagcgt ca
//
