LOCUS       sequence-496500-        8628 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  Vector encoding human codon-optimized engineered DelIscB nickase 
            fusion with APOBEC3A and UGI 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 8628)
  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  JOURNAL   Unpublished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 8628)
  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            "Unpublished"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: ICBE
FEATURES             Location/Qualifiers
     source          1..8628
                     /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..1200
                     /label=chicken beta-actin promoter
     intron          1201..1428
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     CDS             3462..3509
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3540..3788
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3819..4067
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4122..4142
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4170..4187)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4176..4383
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4384..4632
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4384..4404
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4555..4574
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        4984..5287
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5288..5491
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5442..5462
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             5520..6230
                     /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(5665..5683)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     rep_origin      6785..7240
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6872..6891)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7082..7103
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7257..7276)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7376..7398
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7436..7454)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7522..7626
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7627..7695,7696..8487)
                     /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(7845..7864)
                     /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 ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg
     1081 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
     1141 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
     1201 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
     1261 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
     1321 ccgggctgta attagctgag caagaggtaa gggtttaagg gatggttggt tggtggggta
     1381 ttaatgttta attacctgga gcacctgcct gaaatcactt tttttcaggt tggaccggtg
     1441 ccaccatgag cagagtgctg gtggtggatg ctgataggcg ccctctggcc ccttgcaccc
     1501 ctcggagagc cagactgctg ctgaattctg gcaaagctgc cgtgctgaga cggtaccctt
     1561 tcaccatcat cctgaagcag agctacccca ccgccagccc tagacccgtg cggctgaagc
     1621 tggcccccgg aagcaagacc accggcatcg ccgtggtgac agaggccaca ggagaagtgg
     1681 tgtgggccgc tgagctgcag cacagaggcc agctgatcaa gaacgccctg gaaaagagaa
     1741 gagccctgcg gcgcagccgg cgaaaccgga agacacggta tagacctgcc agattcctga
     1801 atagaaagag aacaggacct ccactgctga gcagcgccga cactgtgtct accctgggca
     1861 agtggttgcc cccgagcctg cagcatagga tcgaggtgat catgacctgg gtccacagac
     1921 tgagaagata cctgcccatc accgccattt ctcaggagat cgtgagattc gacatgcaaa
     1981 agatgcagaa ccccgaaatc agcggcgtgg aataccagca gggcaccctc ttcggctacg
     2041 aggtgcggga atacctcctg gacaagtggc ggagacaatg tggctactgc ggcgctaagg
     2101 acaagagact tgaggtcgac cacatcgttc ctcggagcca cggcggcagc gacagagtgt
     2161 ccaacctgac actgtcctgc gagccttgca acaaaagaaa gaaccagagg ccagctgctg
     2221 tgttcctggc taaaaagccc gaggtcctgc agaagctgca gcggcaggcc aaggcccctc
     2281 tgaaggacgc cgccgccgtg aacagcaccc gttacgccct gctggaaaga ctgaaggcca
     2341 ccggcctgcc tgtggaagtg gccagcggcg gaagaaccaa gttcaataga agcaggagac
     2401 agatccccaa gacccactgg ctggatgccg cctgcgtggg cgcttctaca cctgaggtgc
     2461 tgaagtggga ggccgttaga ccactggcca tcaaggccat gggccacggc aaacggcagg
     2521 tagtcaacgt ggacgcctac ggatttcctc ggggcaaggc caaaggcatc cctgtgcacc
     2581 ccttcagaac cggagatatc gtgcgggccg agattccaaa gggcaaatac aagggaacat
     2641 acgtgtccag aatcgccgag acaaccacat ctaagcctct agcaggcttt aagagcaaga
     2701 ccggcaagcg gatccagtgt cacaccaagc acatgacaaa gctgttcaac cgcgacggct
     2761 atggctacgg cttcctgaaa gcccctgaac ctagacagac cgtgatcagc gagtctagcg
     2821 gcagcgagac tcccgggacc tcagagtccg ccacacccga aagtatggag gcatctccag
     2881 caagcggacc aaggcacctg atggaccccc acatcttcac ctctaacttt aacaatggca
     2941 tcggcaggca caagacatac ctgtgctatg aggtggagcg cctggacaat ggcaccagcg
     3001 tgaagatgga tcagcacaga ggcttcctgc acaaccaggc caagaatctg ctgtgcggct
     3061 tctacggccg gcacgcagag ctgagatttc tggacctggt gcctagcctg cagctggatc
     3121 cagcccagat ctatagggtg acctggttca tcagctactc cccatgcttt tccgcgggat
     3181 gtgcaggaga ggtgcgcgcc ttcctgcagg agaacacaca cgtgcggctg agaatctttg
     3241 ccgcccggat cttcgactat gatcctctgt acaaggaggc cctgcagatg ctgagagacg
     3301 caggagccca ggtgtccatc atgacctatg atgagttcaa gcactgctgg gacacatttg
     3361 tggatcacca gggctgtccc tttcagcctt gggacggact ggatgagcac tcccaggccc
     3421 tgtctggcag gctgagggcc atcctgcaga accagggcaa taaaaggccg gcggccacga
     3481 agaaggccgg ccaggcaaag aagaaaaaga gcggcgggag cggcgggagc ggggggagca
     3541 ctaatctgtc agatattatt gaaaaggaaa ccggtaagca actggttatc caggaatcca
     3601 tcctcatgct cccagaggag gtggaagaag tcattgggaa caagccggaa agcgatatac
     3661 tcgtgcacac cgcctacgac gagagcaccg acgagaatgt catgcttctg actagcgacg
     3721 cccctgaata caagccttgg gctctggtca tacaggatag caacggtgag aacaagatta
     3781 agatgctcag cggaggatcc ggaggatctg gaggcagcac caacctgtct gacatcatcg
     3841 agaaggagac aggcaagcag ctggtcatcc aggagagcat cctgatgctg ccagaggagg
     3901 tggaggaagt gatcggcaac aagcccgagt ccgatatcct ggtgcacacc gcctacgacg
     3961 agagcacaga tgagaatgtg atgctgctga cctccgacgc ccccgagtat aagccttggg
     4021 ccctggtcat ccaggattct aacggcgaga ataagatcaa gatgctgtct ggtggttctt
     4081 ctggcggctc aaagagaacc gccgacggca gcgaatccga gcccaagaag aagaggaaag
     4141 tctaagaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     4201 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     4261 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     4321 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg
     4381 ggagagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     4441 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     4501 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     4561 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     4621 ggacgaaaca ccggggagac cgagagaggg tctcagttgt agcctccact cattgaaaaa
     4681 tgagtgcggg aaaccgcagc tacaaccaga ctaagttctt tgggaactac gttattgtcg
     4741 tcatcatacc cacgggtgcg tgccagcctg tggctctatg gtcgtgcatt aaacagggac
     4801 tttggagggt gcccagtgtg tacggcgtca aaagcggcga taacattgtc gaggcaaact
     4861 ttacgtgacg gcgaaagccc gatcgctagc tttaaggcta attttttatt tttttgcggc
     4921 cgctagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     4981 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     5041 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     5101 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     5161 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     5221 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     5281 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     5341 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     5401 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     5461 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagca
     5521 tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg
     5581 tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     5641 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct
     5701 tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc
     5761 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg
     5821 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
     5881 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
     5941 tgcagaagaa aaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg
     6001 ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg
     6061 aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca
     6121 acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac
     6181 gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtaa ggatccggga
     6241 tgcagaaatt gatgatctat taaacaataa agatgtccac taaaatggaa gtttttcctg
     6301 tcatactttg ttaagaaggg tgagaacaga gtacctacat tttgaatgga aggattggag
     6361 ctacgggggt gggggtgggg tgggattaga taaatgcctg ctctttactg aaggctcttt
     6421 actattgctt tatgataatg tttcatagtt ggatatcata atttaaacaa gcaaaaccaa
     6481 attaagggcc agctcattcc tcccactcat gatctataga tctatagatc tctcgtggga
     6541 tcattgtttt tctcttgatt cccactttgt ggttctaagt actgtggttt ccaaatgtgt
     6601 cagtttcata gcctgaagaa cgagatcagc agcctctgtt ccacatacac ttcattctca
     6661 gtattgtttt gccaagttct aattccatca gaagctggtc gacctgcagg ggcgcctgat
     6721 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     6781 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     6841 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     6901 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     6961 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     7021 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     7081 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     7141 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     7201 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     7261 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     7321 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     7381 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     7441 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     7501 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     7561 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     7621 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     7681 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     7741 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     7801 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     7861 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     7921 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     7981 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     8041 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     8101 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     8161 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     8221 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     8281 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     8341 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     8401 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     8461 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     8521 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     8581 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtca
//
