LOCUS       sequence-453972-        6849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector encoding human codon-optimized enhanced-activity nOgeuIscB 
            fusion with TadA8e and HMG-D domain in N terminus driven by CMV 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6849)
  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 6849)
  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-N-HMG-D
FEATURES             Location/Qualifiers
     source          1..6849
                     /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             3019..3039
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3049..3105
                     /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(3106..3108,3109..3111,3112..3822)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3106..3108,3109..3111,3112..3822)
                     /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(3151..3172)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3412..3431)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3759..3780
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3835..3855
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3864..3881
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3904..3921)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3910..4134
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4205..4221)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4205..4221)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4218..4240)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4229..4245
                     /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(4253..4259,4260..4277,4278..4283))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4298..4319
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4436..4453)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4607..5195)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4687..4706)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5366..6157,6158..6226))
                     /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     5989..6008
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6227..6331)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6410..6429)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6601..6849,1..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 aagtcatgag cgacaaaccg
      481 aaacggccgc tttctgctta catgctctgg ctgaactcag cacgagagag catcaagaga
      541 gaaaatcccg gcattaaagt cactgaggtg gctaaacgcg gtggcgaact ttggagggca
      601 atgaaagata agtccgagtg ggaggccaag gcagcgaaag cgaaggatga ctatgaccgc
      661 gccgtaaaag agtttgaggc aaatgggggt agcagcgcgg caaatggagg cggcgcgaag
      721 aaaagggcaa agcctgctaa gaaagttgct aagaagtcta aaaaagaaga atcagatgag
      781 gatgatgacg acgagtctga gtccggagga tctagcggag gctcctctgg ctctgagaca
      841 cctggcacaa gcgagagcgc aacacctgaa agcagcgggg gcagcagcgg ggggtcatct
      901 gaggtggagt tttcccacga gtactggatg agacatgccc tgaccctggc caagagggca
      961 cgggatgaga gggaggtgcc tgtgggagcc gtgctggtgc tgaacaatag agtgatcggc
     1021 gagggctgga acagagccat cggcctgcac gacccaacag cccatgccga aattatggcc
     1081 ctgagacagg gcggcctggt catgcagaac tacagactga ttgacgccac cctgtacgtg
     1141 acattcgagc cttgcgtgat gtgcgccggc gccatgatcc actctaggat cggccgcgtg
     1201 gtgtttggcg tgaggaactc aaaaagaggc gccgcaggct ccctgatgaa cgtgctgaac
     1261 taccccggca tgaatcaccg cgtcgaaatt accgagggaa tcctggcaga tgaatgtgcc
     1321 gccctgctgt gcgatttcta tcggatgcct agacaggtgt tcaatgctca gaagaaggcc
     1381 cagagctcca tcaactccgg aggatctagc ggaggctcct ctggctctga gacacctggc
     1441 acaagcgaga gcgcaacacc tgaaagcagc gggggcagca gcggggggtc aatggccgtg
     1501 gtctacgtga tctctaagtc cggcaaacca ctgatgccca ccaccagatg cggccacgtg
     1561 cggatcttgc tcaaggaagg caaggctaga gtggtggaaa gaaagccctt caccatccag
     1621 ctgacctacg agtctgccga ggaaacccag cctctggtgc tgggcattga ccctggcaga
     1681 accaatatcg gtatgagcgt ggtgaccgag agcggagaga gcgtgttcaa cgcccaaatc
     1741 agaacaagaa acaaggacgt gcctaagctg atgaagagaa gaaagcagta taggatggcc
     1801 cacagaaggc tgaagcggcg gtgcaaaaga cggcggagag ccaaggctgc cggcaccgcc
     1861 ttcgaggagg gcgaaaagca aagactgctg cctggctgct tcaagcctat tacatgcaag
     1921 agcatccgga acaaggaagc cagattcaac aaccgcaagc ggcctagagg atggctgaca
     1981 cctaccgcca atcacctgct ggtcacccac ctgaacgtgg ttaagaaggt gcagaagatc
     2041 ctgcccgtgg ccaaggtggt gctggagctg aatcggttca gctttatggc catgaacaac
     2101 cccaaggttc agagatggca gtaccagcgg ggccctctgt acggcaaagg cagcgtggaa
     2161 gaggccgtgt ccatgcagca ggatggacac tgcctgtttt gtaaacacgg catcgaccac
     2221 taccaccacg tggtccccag aagaaagaac ggcagcgaga cactggaaaa cagagtgggc
     2281 ctttgtgaag aacaccatag actggtgcac acagataagg agtgggaggc caacctggcc
     2341 agcaagaagt ctggaatgaa caagaaatac cacgccctgt ctgtgctgaa ccagatcatc
     2401 ccctacctgg ctgatcagct ggccgatatg ttccccggaa atttctgcgt gacaagcggc
     2461 caagacacct atctgtttcg ggaagagcat ggcatcccta aggacgccta cctggacgcc
     2521 tactgcatcg cctgcagcgc actgaccgac gccaagaagg tgagcagccc taagggcaga
     2581 ccatacatgg tgcaccagtt cagaaggcat gatagacagg cctgtcacaa ggccaatctg
     2641 aaccggagct actacatggg cggcaagctc gttgccacca accggcacaa agctatggac
     2701 cagaaaactg acagcctgga agagtacaga gccgctcaca gcgccgctga cgtgtctaaa
     2761 ctgaccgtga agcacccttc tgctcagtac aaggatatga gcagaatcat gcctggcagc
     2821 atcctggtga gcggcgaggg caaactgttc acactgagca gatctgaggg aagaaacaaa
     2881 ggccaggtga actacttcgt gtccaccgag ggcatcaagt actgggccag aaagtgccag
     2941 tatctgcgga acaacggcgg actgcagatc tacgtgtctg gcggctcaaa aagaaccgcc
     3001 gacggcagcg aattcgagcc caagaagaag aggaaagtcg gaagcggagc tactaacttc
     3061 agcctgctga agcaggctgg agacgtggag gagaaccctg gacctatggt gagcaagggc
     3121 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     3181 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     3241 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     3301 acctatggag tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     3361 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     3421 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     3481 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     3541 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     3601 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     3661 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     3721 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     3781 accgccgccg ggatcactct cggcatggac gagctgtaca agtctggtgg ttctcccaag
     3841 aagaagagga aagtctaacc ggtcatcatc accatcacca ttgagtttaa acccgctgat
     3901 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3961 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     4021 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     4081 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     4141 aggcggaaag aaccagctgg ggctcgatac cgtcgacctc tagctagagc ttggcgtaat
     4201 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4261 gagccggaag cataaagtgt aaagcctagg gtgcctaatg agtgagctaa ctcacattaa
     4321 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4381 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4441 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4501 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4561 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4621 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4681 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4741 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4801 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4861 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4921 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4981 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5041 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5101 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5161 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5221 ggtctgacac tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5281 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5341 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5401 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5461 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5521 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5581 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5641 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5701 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5761 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5821 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5881 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5941 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6001 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6061 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6121 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6181 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6241 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6301 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6361 tcgacggatc gggagatcga tctcccgatc ccctagggtc gactctcagt acaatctgct
     6421 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     6481 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     6541 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
     6601 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     6661 catatattga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     6721 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     6781 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     6841 aagtgtatc
//
