LOCUS       sequence-496504-        8326 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  Vector encoding human codon-optimized engineered DelIscB fused with 
            T5E at C-terminal 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 8326)
  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  JOURNAL   Unpublished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 8326)
  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            "Unpublished"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: enDelIscB-T5E
FEATURES             Location/Qualifiers
     source          1..8326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..293
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        295..568
                     /label=chicken beta-actin promoter
     intron          569..796
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3163..3210
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(3238..3255)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3244..3451
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3452..3700
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3452..3472
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3623..3642
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        4052..4355
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4356..4559
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4510..4530
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4588..5298
                     /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(4733..4751)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     rep_origin      5853..6308
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5940..5959)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6150..6171
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6325..6344)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6444..6466
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6504..6522)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6590..6694
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6695..6763,6764..7555)
                     /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(6913..6932)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7726..8314
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8215..8234
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
       61 gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      121 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      181 ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat
      241 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
      301 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
      361 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggcgcgcg
      421 ccaggcgggg gggggggggg gggggggggg gggggggggg cggagaggtg cggcggcagc
      481 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc
      541 ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc
      601 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
      661 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagctgagca agaggtaagg
      721 gtttaaggga tggttggttg gtggggtatt aatgtttaat tacctggagc acctgcctga
      781 aatcactttt tttcaggttg gaccggtgcc accatgagca gagtgctggt ggtggatgct
      841 gataggcgcc ctctggcccc ttgcacccct cggagagcca gactgctgct gaattctggc
      901 aaagctgccg tgctgagacg gtaccctttc accatcatcc tgaagcagag ctaccccacc
      961 gccagcccta gacccgtgcg gctgaagctg gaccccggaa gcaagaccac cggcatcgcc
     1021 gtggtgacag aggccacagg agaagtggtg tgggccgctg agctgcagca cagaggccag
     1081 ctgatcaaga acgccctgga aaagagaaga gccctgcggc gcagccggcg aaaccggaag
     1141 acacggtata gacctgccag attcctgaat agaaagagaa caggacctcc actgctgagc
     1201 agcgccgaca ctgtgtctac cctgggcaag tggttgcccc cgagcctgca gcataggatc
     1261 gaggtgatca tgacctgggt ccacagactg agaagatacc tgcccatcac cgccatttct
     1321 caggagatcg tgagattcga catgcaaaag atgcagaacc ccgaaatcag cggcgtggaa
     1381 taccagcagg gcaccctctt cggctacgag gtgcgggaat acctcctgga caagtggcgg
     1441 agacaatgtg gctactgcgg cgctaaggac aagagacttg aggtcgacca catcgttcct
     1501 cggagccacg gcggcagcga cagagtgtcc aacctgacac tgtcctgcga gccttgcaac
     1561 aaaagaaaga accagaggcc agctgctgtg ttcctggcta aaaagcccga ggtcctgcag
     1621 aagctgcagc ggcaggccaa ggcccctctg aaggacgccg ccgccgtgaa cagcacccgt
     1681 tacgccctgc tggaaagact gaaggccacc ggcctgcctg tggaagtggc cagcggcgga
     1741 agaaccaagt tcaatagaag caggagacag atccccaaga cccactggct ggatgccgcc
     1801 tgcgtgggcg cttctacacc tgaggtgctg aagtgggagg ccgttagacc actggccatc
     1861 aaggccatgg gccacggcaa acggcaggtg gtcaacgtgg acgcctacgg atttcctcgg
     1921 ggcaaggcca aaggcatccc tgtgcacccc ttcagaaccg gagatatcgt gcgggccgag
     1981 attccaaagg gcaaatacaa gggaacatac gtgtccagaa tcgccgagac aaccacatct
     2041 aagcctctag caggctttaa gagcaagacc ggcaagcgga tccagtgtca caccaagcac
     2101 atgacaaagc tgttcaaccg cgacggctat ggctacggct tcctgaaagc ccctgaacct
     2161 agacagaccg tgatcagcga gtctaagctt tccggaggat ctagcggagg ctcctctggc
     2221 agcgagacac caggaacaag cgagtcagca acaccagaga gcagtggcgg cagcagcggc
     2281 ggcagcagta tgagcaagtc ctggggcaag ttcatcgagg aagaggaagc tgaaatggcc
     2341 tctagacgca acctgatgat cgtggatggc accaacctcg gcttcaggtt caagcacaac
     2401 aactccaaga aacctttcgc cagcagctac gtgtctacca tccagagcct ggccaagagc
     2461 tatagcgcca gaaccacaat cgtgctcggc gataagggca aaagcgtgtt cagactggaa
     2521 catctgcctg agtacaaggg caaccgggac gagaagtacg cccagcggac cgaggaagag
     2581 aaggctctgg atgagcagtt tttcgagtac ctgaaagacg ccttcgagct ttgtaaaaca
     2641 acatttccta cattcacaat cagaggagtg gaagccgatg atatggccgc ttatatcgtg
     2701 aagctgatcg gccacctgta cgaccacgtg tggctgatct ctacagatgg tgactgggac
     2761 accctgctga ccgacaaggt gtccagattc agcttcacca ccagaagaga gtaccacctg
     2821 agagatatgt acgagcacca caatgtggac gacgtggagc aatttatcag cctgaaggcc
     2881 attatgggcg acctgggcga caacatccgg ggcgttgaag gcatcggcgc taaaagaggc
     2941 tacaacatca tccgggagtt cggcaatgtg ctggacatca tcgaccagct gcccctgcca
     3001 ggcaagcaga aatacatcca gaacctgaac gccagcgagg agctgctgtt cagaaatctg
     3061 attctggtgg acctgcctac ctactgcgtg gatgccatcg ccgccgtggg ccaggacgtg
     3121 ctggacaagt ttaccaagga catcctggaa atcgccgagc agaaaaggcc ggcggccacg
     3181 aagaaggccg gccaggcaaa gaagaagaag taagaattcc tagagctcgc tgatcagcct
     3241 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3301 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3361 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3421 attgggaaga gaatagcagg catgctgggg agagggccta tttcccatga ttccttcata
     3481 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     3541 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     3601 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     3661 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggggagaccg agagagggtc
     3721 tcagttgtag cctccactca ttgaaaaatg agtgcgggaa accgcagcta caaccagact
     3781 aagttctttg ggaactacgt tattgtcgtc atcataccca cgggtgcgtg ccagcctgtg
     3841 gctctatggt cgtgcattaa acagggactt tggagggtgc ccagtgtgta cggcgtcaaa
     3901 agcggcgata acattgtcga ggcaaacttt acgtgacggc gaaagcccga tcgctagctt
     3961 taaggctaat tttttatttt tttgcggccg ctagttatta atagtaatca attacggggt
     4021 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     4081 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     4141 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     4201 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     4261 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     4321 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     4381 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     4441 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     4501 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg
     4561 gtttagtgaa ccgtcagatc cgctagcatg gtgagcaagg gcgaggagga taacatggcc
     4621 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     4681 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     4741 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     4801 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     4861 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     4921 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     4981 accaacttcc cctccgacgg ccccgtaatg cagaagaaaa ccatgggctg ggaggcctcc
     5041 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     5101 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     5161 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     5221 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     5281 gacgagctgt acaagtaagg atccgggatg cagaaattga tgatctatta aacaataaag
     5341 atgtccacta aaatggaagt ttttcctgtc atactttgtt aagaagggtg agaacagagt
     5401 acctacattt tgaatggaag gattggagct acgggggtgg gggtggggtg ggattagata
     5461 aatgcctgct ctttactgaa ggctctttac tattgcttta tgataatgtt tcatagttgg
     5521 atatcataat ttaaacaagc aaaaccaaat taagggccag ctcattcctc ccactcatga
     5581 tctatagatc tatagatctc tcgtgggatc attgtttttc tcttgattcc cactttgtgg
     5641 ttctaagtac tgtggtttcc aaatgtgtca gtttcatagc ctgaagaacg agatcagcag
     5701 cctctgttcc acatacactt cattctcagt attgttttgc caagttctaa ttccatcaga
     5761 agctggtcga cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     5821 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     5881 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc
     5941 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     6001 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     6061 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     6121 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     6181 caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg
     6241 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     6301 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     6361 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     6421 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     6481 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     6541 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     6601 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     6661 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     6721 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     6781 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     6841 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     6901 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     6961 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     7021 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     7081 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     7141 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     7201 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     7261 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     7321 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     7381 tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc
     7441 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     7501 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     7561 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     7621 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     7681 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     7741 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     7801 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     7861 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     7921 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     7981 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     8041 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     8101 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     8161 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     8221 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     8281 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaac
//
