LOCUS       sequence-453978-        7478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector encoding human codon-optimized enhanced-activity nOgeuIscB 
            fusion with two copies of UGI and hA3A* variant and HMG-D domain was
            inserted between hA3A* and eIscBn driven by CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7478)
  AUTHORS   Xue N, Hong D, Zhang D, Wang Q, Zhang S, Yang L, Chen X, Li Y, Han 
            H, Hu C, Liu M, Song G, Guan Y, Wang L, Zhu Y, Li D
  TITLE     Engineering IscB to develop highly efficient miniature editing tools
            in mammalian cells and embryos.
  JOURNAL   Mol Cell. 2024 Aug 22;84(16):3128-3140.e4. doi: 
            10.1016/j.molcel.2024.07.007. Epub 2024 Aug 2.
  PUBMED    39096898
REFERENCE   2  (bases 1 to 7478)
  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"; doi: 
            "10.1016/j.molcel.2024.07.007"; journalName: "Mol Cell"; date: 
            "2024-08-22- 22"; volume: "84"; issue: "16"; pages: "3128-3140.e4"
COMMENT     Sequence Label: hA3A*-eIscBn-D
FEATURES             Location/Qualifiers
     source          1..7478
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             417..437
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3000..3047
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3078..3326
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3357..3605
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3648..3668
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3678..3734
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(3735..3737,3738..3740,3741..4451)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3735..3737,3738..3740,3741..4451)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3780..3801)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4041..4060)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4388..4409
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4464..4484
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4493..4510
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4533..4550)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4539..4763
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4834..4850)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4834..4850)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4847..4869)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4858..4874
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4882..4888,4889..4906,4907..4912))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4927..4948
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5065..5082)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5236..5824)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5316..5335)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5995..6786,6787..6855))
                     /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     6618..6637
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6856..6960)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7039..7058)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7230..7478,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgccacc atgaagagga ccgccgatgg ctctgagttc gagagcccca
      421 agaagaagcg gaaggtgatg gaggcatctc cagcaagcgg accaaggcac ctgatggacc
      481 cccacatctt cacctctaac tttaacaatg gcatcggcag gcacaagaca tacctgtgct
      541 atgaggtgga gcgcctggac aatggcacca gcgtgaagat ggatcagcac agaggcttcc
      601 tgcacaacca ggccaagaat ctgctgtgcg gcttctacgg ccggcacgca gagctgagat
      661 ttctggacct ggtgcctagc ctgcagctgg atccagccca gatctatagg gtgacctggt
      721 tcatcagcta ctccccatgc ttttccgcgg gatgtgcagg agaggtgcgc gccttcctgc
      781 aggagaacac acacgtgcgg ctgagaatct ttgccgcccg gatcttcgac tatgatcctc
      841 tgtacaagga ggccctgcag atgctgagag acgcaggagc ccaggtgtcc atcatgacct
      901 atgatgagtt caagcactgc tgggacacat ttgtggatca ccagggctgt ccctttcagc
      961 cttgggacgg actggatgag cactcccagg ccctgtctgg caggctgagg gccatcctgc
     1021 agaaccaggg caattctgga ggatctagcg gaggatcctc tggcagcgag acaccaggaa
     1081 caagcgagtc agcaacacca gagagcagtg gcggcagcag cggcggcagc atgagcgaca
     1141 aaccgaaacg gccgctttct gcttacatgc tctggctgaa ctcagcacga gagagcatca
     1201 agagagaaaa tcccggcatt aaagtcactg aggtggctaa acgcggtggc gaactttgga
     1261 gggcaatgaa agataagtcc gagtgggagg ccaaggcagc gaaagcgaag gatgactatg
     1321 accgcgccgt aaaagagttt gaggcaaatg ggggtagcag cgcggcaaat ggaggcggcg
     1381 cgaagaaaag ggcaaagcct gctaagaaag ttgctaagaa gtctaaaaaa gaagaatcag
     1441 atgaggatga tgacgacgag tctgagagcg gcagcgagac tcccgggacc tcagagtccg
     1501 ccacacccga aagtatggcc gtggtctacg tgatctctaa gtccggcaaa ccactgatgc
     1561 ccaccaccag atgcggccac gtgcggatct tgctcaagga aggcaaggct agagtggtgg
     1621 aaagaaagcc cttcaccatc cagctgacct acgagtctgc cgaggaaacc cagcctctgg
     1681 tgctgggcat tgaccctggc agaaccaata tcggtatgag cgtggtgacc gagagcggag
     1741 agagcgtgtt caacgcccaa atcagaacaa gaaacaagga cgtgcctaag ctgatgaaga
     1801 gaagaaagca gtataggatg gcccacagaa ggctgaagcg gcggtgcaaa agacggcgga
     1861 gagccaaggc tgccggcacc gccttcgagg agggcgaaaa gcaaagactg ctgcctggct
     1921 gcttcaagcc tattacatgc aagagcatcc ggaacaagga agccagattc aacaaccgca
     1981 agcggcctag aggatggctg acacctaccg ccaatcacct gctggtcacc cacctgaacg
     2041 tggttaagaa ggtgcagaag atcctgcccg tggccaaggt ggtgctggag ctgaatcggt
     2101 tcagctttat ggccatgaac aaccccaagg ttcagagatg gcagtaccag cggggccctc
     2161 tgtacggcaa aggcagcgtg gaagaggccg tgtccatgca gcaggatgga cactgcctgt
     2221 tttgtaaaca cggcatcgac cactaccacc acgtggtccc cagaagaaag aacggcagcg
     2281 agacactgga aaacagagtg ggcctttgtg aagaacacca tagactggtg cacacagata
     2341 aggagtggga ggccaacctg gccagcaaga agtctggaat gaacaagaaa taccacgccc
     2401 tgtctgtgct gaaccagatc atcccctacc tggctgatca gctggccgat atgttccccg
     2461 gaaatttctg cgtgacaagc ggccaagaca cctatctgtt tcgggaagag catggcatcc
     2521 ctaaggacgc ctacctggac gcctactgca tcgcctgcag cgcactgacc gacgccaaga
     2581 aggtgagcag ccctaagggc agaccataca tggtgcacca gttcagaagg catgatagac
     2641 aggcctgtca caaggccaat ctgaaccgga gctactacat gggcggcaag ctcgttgcca
     2701 ccaaccggca caaagctatg gaccagaaaa ctgacagcct ggaagagtac agagccgctc
     2761 acagcgccgc tgacgtgtct aaactgaccg tgaagcaccc ttctgctcag tacaaggata
     2821 tgagcagaat catgcctggc agcatcctgg tgagcggcga gggcaaactg ttcacactga
     2881 gcagatctga gggaagaaac aaaggccagg tgaactactt cgtgtccacc gagggcatca
     2941 agtactgggc cagaaagtgc cagtatctgc ggaacaacgg cggactgcag atctacgtga
     3001 aaaggccggc ggccacgaag aaggccggcc aggcaaagaa gaaaaagagc ggcgggagcg
     3061 gcgggagcgg ggggagcact aatctgagcg acatcattga gaaggagact gggaaacagc
     3121 tggtcattca ggagtccatc ctgatgctgc ctgaggaggt ggaggaagtg atcggcaaca
     3181 agccagagtc tgacatcctg gtgcacaccg cctacgacga gtccacagat gagaatgtga
     3241 tgctgctgac ctctgacgcc cccgagtata agccttgggc cctggtcatc caggattcta
     3301 acggcgagaa taagatcaag atgctgagcg gaggatccgg aggatctgga ggcagcacca
     3361 acctgtctga catcatcgag aaggagacag gcaagcagct ggtcatccag gagagcatcc
     3421 tgatgctgcc cgaagaagtc gaagaagtga tcggaaacaa gcctgagagc gatatcctgg
     3481 tccataccgc ctacgacgag agtaccgacg aaaatgtgat gctgctgaca tccgacgccc
     3541 cagagtataa gccctgggct ctggtcatcc aggattccaa cggagagaac aaaatcaaaa
     3601 tgctgtctgg cggctcaaaa agaaccgccg acggcagcga attcgagccc aagaagaaga
     3661 ggaaagtcgg aagcggagct actaacttca gcctgctgaa gcaggctgga gacgtggagg
     3721 agaaccctgg acctatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     3781 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     3841 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     3901 tgccctggcc caccctcgtg accaccctga cctatggagt gcagtgcttc agccgctacc
     3961 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     4021 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     4081 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     4141 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     4201 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     4261 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     4321 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     4381 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     4441 agctgtacaa gtctggtggt tctcccaaga agaagaggaa agtctaaccg gtcatcatca
     4501 ccatcaccat tgagtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     4561 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     4621 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     4681 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     4741 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctcgatacc
     4801 gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     4861 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctaggg
     4921 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4981 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5041 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5101 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5161 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5221 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5281 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5341 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5401 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     5461 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5521 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5581 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5641 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     5701 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5761 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5821 tcaagaagat cctttgatct tttctacggg gtctgacact cagtggaacg aaaactcacg
     5881 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5941 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     6001 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6061 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6121 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6181 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6241 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6301 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6361 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6421 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6481 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6541 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6601 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6661 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6721 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6781 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6841 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6901 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6961 cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatcgat ctcccgatcc
     7021 cctagggtcg actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     7081 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     7141 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     7201 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     7261 atcaattacg gggtcattag ttcatagccc atatattgag ttccgcgtta cataacttac
     7321 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     7381 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     7441 acggtaaact gcccacttgg cagtacatca agtgtatc
//
