LOCUS       sequence-453981-        7046 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 driven by CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7046)
  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 7046)
  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: eiCBE
FEATURES             Location/Qualifiers
     source          1..7046
                     /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             2568..2615
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             2646..2894
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             2925..3173
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3216..3236
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3246..3302
                     /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(3303..3305,3306..3308,3309..4019)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3303..3305,3306..3308,3309..4019)
                     /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(3348..3369)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3609..3628)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3956..3977
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4032..4052
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4061..4078
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4101..4118)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4107..4331
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4402..4418)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4402..4418)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4415..4437)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4426..4442
                     /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(4450..4456,4457..4474,4475..4480))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4495..4516
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4633..4650)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4804..5392)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4884..4903)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5563..6354,6355..6423))
                     /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     6186..6205
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6424..6528)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6607..6626)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6798..7046,1..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 caatagcggc agcgagactc ccgggacctc agagtccgcc acacccgaaa
     1081 gtatggccgt ggtctacgtg atctctaagt ccggcaaacc actgatgccc accaccagat
     1141 gcggccacgt gcggatcttg ctcaaggaag gcaaggctag agtggtggaa agaaagccct
     1201 tcaccatcca gctgacctac gagtctgccg aggaaaccca gcctctggtg ctgggcattg
     1261 accctggcag aaccaatatc ggtatgagcg tggtgaccga gagcggagag agcgtgttca
     1321 acgcccaaat cagaacaaga aacaaggacg tgcctaagct gatgaagaga agaaagcagt
     1381 ataggatggc ccacagaagg ctgaagcggc ggtgcaaaag acggcggaga gccaaggctg
     1441 ccggcaccgc cttcgaggag ggcgaaaagc aaagactgct gcctggctgc ttcaagccta
     1501 ttacatgcaa gagcatccgg aacaaggaag ccagattcaa caaccgcaag cggcctagag
     1561 gatggctgac acctaccgcc aatcacctgc tggtcaccca cctgaacgtg gttaagaagg
     1621 tgcagaagat cctgcccgtg gccaaggtgg tgctggagct gaatcggttc agctttatgg
     1681 ccatgaacaa ccccaaggtt cagagatggc agtaccagcg gggccctctg tacggcaaag
     1741 gcagcgtgga agaggccgtg tccatgcagc aggatggaca ctgcctgttt tgtaaacacg
     1801 gcatcgacca ctaccaccac gtggtcccca gaagaaagaa cggcagcgag acactggaaa
     1861 acagagtggg cctttgtgaa gaacaccata gactggtgca cacagataag gagtgggagg
     1921 ccaacctggc cagcaagaag tctggaatga acaagaaata ccacgccctg tctgtgctga
     1981 accagatcat cccctacctg gctgatcagc tggccgatat gttccccgga aatttctgcg
     2041 tgacaagcgg ccaagacacc tatctgtttc gggaagagca tggcatccct aaggacgcct
     2101 acctggacgc ctactgcatc gcctgcagcg cactgaccga cgccaagaag gtgagcagcc
     2161 ctaagggcag accatacatg gtgcaccagt tcagaaggca tgatagacag gcctgtcaca
     2221 aggccaatct gaaccggagc tactacatgg gcggcaagct cgttgccacc aaccggcaca
     2281 aagctatgga ccagaaaact gacagcctgg aagagtacag agccgctcac agcgccgctg
     2341 acgtgtctaa actgaccgtg aagcaccctt ctgctcagta caaggatatg agcagaatca
     2401 tgcctggcag catcctggtg agcggcgagg gcaaactgtt cacactgagc agatctgagg
     2461 gaagaaacaa aggccaggtg aactacttcg tgtccaccga gggcatcaag tactgggcca
     2521 gaaagtgcca gtatctgcgg aacaacggcg gactgcagat ctacgtgaaa aggccggcgg
     2581 ccacgaagaa ggccggccag gcaaagaaga aaaagagcgg cgggagcggc gggagcgggg
     2641 ggagcactaa tctgagcgac atcattgaga aggagactgg gaaacagctg gtcattcagg
     2701 agtccatcct gatgctgcct gaggaggtgg aggaagtgat cggcaacaag ccagagtctg
     2761 acatcctggt gcacaccgcc tacgacgagt ccacagatga gaatgtgatg ctgctgacct
     2821 ctgacgcccc cgagtataag ccttgggccc tggtcatcca ggattctaac ggcgagaata
     2881 agatcaagat gctgagcgga ggatccggag gatctggagg cagcaccaac ctgtctgaca
     2941 tcatcgagaa ggagacaggc aagcagctgg tcatccagga gagcatcctg atgctgcccg
     3001 aagaagtcga agaagtgatc ggaaacaagc ctgagagcga tatcctggtc cataccgcct
     3061 acgacgagag taccgacgaa aatgtgatgc tgctgacatc cgacgcccca gagtataagc
     3121 cctgggctct ggtcatccag gattccaacg gagagaacaa aatcaaaatg ctgtctggcg
     3181 gctcaaaaag aaccgccgac ggcagcgaat tcgagcccaa gaagaagagg aaagtcggaa
     3241 gcggagctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac
     3301 ctatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     3361 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     3421 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     3481 ccctcgtgac caccctgacc tatggagtgc agtgcttcag ccgctacccc gaccacatga
     3541 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     3601 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     3661 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     3721 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     3781 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     3841 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     3901 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     3961 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     4021 ctggtggttc tcccaagaag aagaggaaag tctaaccggt catcatcacc atcaccattg
     4081 agtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     4141 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     4201 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     4261 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     4321 gggctctatg gcttctgagg cggaaagaac cagctggggc tcgataccgt cgacctctag
     4381 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4441 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt
     4501 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4561 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4621 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4681 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4741 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4801 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4861 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4921 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4981 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5041 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5101 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5161 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5221 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5281 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5341 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5401 tttgatcttt tctacggggt ctgacactca gtggaacgaa aactcacgtt aagggatttt
     5461 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5521 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5581 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5641 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5701 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5761 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5821 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5881 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5941 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6001 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6061 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6121 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6181 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6241 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6301 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6361 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6421 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6481 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6541 aaaagtgcca cctgacgtcg acggatcggg agatcgatct cccgatcccc tagggtcgac
     6601 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     6661 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     6721 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     6781 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
     6841 gtcattagtt catagcccat atattgagtt ccgcgttaca taacttacgg taaatggccc
     6901 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     6961 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     7021 ccacttggca gtacatcaag tgtatc
//
