LOCUS       sequence-453983-        6417 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector encoding human codon-optimized enhanced-activity nOgeuIscB 
            fusion with TadA8e driven by CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6417)
  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 6417)
  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: TadA-8e-eIscBn
FEATURES             Location/Qualifiers
     source          1..6417
                     /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"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2587..2607
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2617..2673
                     /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(2674..2676,2677..2679,2680..3390)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2674..2676,2677..2679,2680..3390)
                     /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(2719..2740)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2980..2999)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3327..3348
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3403..3423
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3432..3449
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3472..3489)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3478..3702
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3773..3789)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3773..3789)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3786..3808)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3797..3813
                     /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(3821..3827,3828..3845,3846..3851))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3866..3887
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4004..4021)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4175..4763)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4255..4274)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4934..5725,5726..5794))
                     /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     5557..5576
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5795..5899)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5978..5997)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6169..6417,1..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 ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga ggtggagttt
      481 tcccacgagt actggatgag acatgccctg accctggcca agagggcacg ggatgagagg
      541 gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac
      601 agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc
      661 ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct
      721 tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg
      781 aggaactcaa aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg
      841 aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc
      901 gatttctatc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc
      961 aactccggag gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc
     1021 gcaacacctg aaagcagcgg gggcagcagc ggggggtcaa tggccgtggt ctacgtgatc
     1081 tctaagtccg gcaaaccact gatgcccacc accagatgcg gccacgtgcg gatcttgctc
     1141 aaggaaggca aggctagagt ggtggaaaga aagcccttca ccatccagct gacctacgag
     1201 tctgccgagg aaacccagcc tctggtgctg ggcattgacc ctggcagaac caatatcggt
     1261 atgagcgtgg tgaccgagag cggagagagc gtgttcaacg cccaaatcag aacaagaaac
     1321 aaggacgtgc ctaagctgat gaagagaaga aagcagtata ggatggccca cagaaggctg
     1381 aagcggcggt gcaaaagacg gcggagagcc aaggctgccg gcaccgcctt cgaggagggc
     1441 gaaaagcaaa gactgctgcc tggctgcttc aagcctatta catgcaagag catccggaac
     1501 aaggaagcca gattcaacaa ccgcaagcgg cctagaggat ggctgacacc taccgccaat
     1561 cacctgctgg tcacccacct gaacgtggtt aagaaggtgc agaagatcct gcccgtggcc
     1621 aaggtggtgc tggagctgaa tcggttcagc tttatggcca tgaacaaccc caaggttcag
     1681 agatggcagt accagcgggg ccctctgtac ggcaaaggca gcgtggaaga ggccgtgtcc
     1741 atgcagcagg atggacactg cctgttttgt aaacacggca tcgaccacta ccaccacgtg
     1801 gtccccagaa gaaagaacgg cagcgagaca ctggaaaaca gagtgggcct ttgtgaagaa
     1861 caccatagac tggtgcacac agataaggag tgggaggcca acctggccag caagaagtct
     1921 ggaatgaaca agaaatacca cgccctgtct gtgctgaacc agatcatccc ctacctggct
     1981 gatcagctgg ccgatatgtt ccccggaaat ttctgcgtga caagcggcca agacacctat
     2041 ctgtttcggg aagagcatgg catccctaag gacgcctacc tggacgccta ctgcatcgcc
     2101 tgcagcgcac tgaccgacgc caagaaggtg agcagcccta agggcagacc atacatggtg
     2161 caccagttca gaaggcatga tagacaggcc tgtcacaagg ccaatctgaa ccggagctac
     2221 tacatgggcg gcaagctcgt tgccaccaac cggcacaaag ctatggacca gaaaactgac
     2281 agcctggaag agtacagagc cgctcacagc gccgctgacg tgtctaaact gaccgtgaag
     2341 cacccttctg ctcagtacaa ggatatgagc agaatcatgc ctggcagcat cctggtgagc
     2401 ggcgagggca aactgttcac actgagcaga tctgagggaa gaaacaaagg ccaggtgaac
     2461 tacttcgtgt ccaccgaggg catcaagtac tgggccagaa agtgccagta tctgcggaac
     2521 aacggcggac tgcagatcta cgtgtctggc ggctcaaaaa gaaccgccga cggcagcgaa
     2581 ttcgagccca agaagaagag gaaagtcgga agcggagcta ctaacttcag cctgctgaag
     2641 caggctggag acgtggagga gaaccctgga cctatggtga gcaagggcga ggagctgttc
     2701 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2761 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     2821 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctatggagtg
     2881 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     2941 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     3001 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     3061 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     3121 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     3181 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     3241 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     3301 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     3361 atcactctcg gcatggacga gctgtacaag tctggtggtt ctcccaagaa gaagaggaaa
     3421 gtctaaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg
     3481 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3541 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3601 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3661 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3721 ccagctgggg ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     3781 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3841 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3901 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3961 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4021 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4081 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4141 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4201 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4261 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4321 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4381 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4441 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4501 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4561 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4621 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4681 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4741 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacactc
     4801 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4861 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4921 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4981 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5041 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5101 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5161 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5221 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5281 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5341 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5401 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5461 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5521 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5581 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5641 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5701 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5761 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5821 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5881 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     5941 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat
     6001 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     6061 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     6121 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
     6181 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatattgagt
     6241 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     6301 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     6361 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatc
//
