LOCUS       sequence-422559-        8349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses 
            NTOM20-SbMVpC-43LK-iLID(139)-NES-NES-SbMVpN-iLID-SbMVseq-bArr2-TevCs
            -P2A-EGFP component in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8349)
  AUTHORS   Cui M, Lee S, Ban SH, Ryu JR, Shen M, Yang SH, Kim JY, Choi SK, Han 
            J, Kim Y, Han K, Lee D, Sun W, Kwon HB, Lee D
  TITLE     A single-component, light-assisted uncaging switch for 
            endoproteolytic release.
  JOURNAL   Nat Chem Biol. 2024 Mar;20(3):353-364. doi: 
            10.1038/s41589-023-01480-6. Epub 2023 Nov 16.
  PUBMED    37973890
REFERENCE   2  (bases 1 to 8349)
  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: 
            "10.1038/s41589-023-01480-6"; journalName: "Nat Chem Biol"; date: 
            "2024-03"; volume: "20"; issue: "3"; pages: "353-364"
COMMENT     Sequence Label: 
            pCMV-NTOM20-SbMVpC-43LK-iLID(139)-NES-NES-SbMVpN-iLID-SbMVseq-bArr2-
            TevCs-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..8349
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..978
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     regulatory      1013..1022
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2102..2128
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             2138..2164
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             4565..4621
                     /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(4631..4633,4634..5347)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(4631..4633,4634..5344)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4673..4694)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4934..4953)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5281..5302
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(5356..5375)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5357..5375)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    5380..5514
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5404..5423)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5458..5477
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5627..5647)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5627..5645)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5666..5682)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5666..5682)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5679..5701)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5690..5706
                     /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(5714..5720,5721..5738,5739..5744))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5759..5780
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5897..5914)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6067..6655)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6147..6166)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6826..7617,7618..7686))
                     /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     7449..7468
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7687..7791)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7817..8272)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     8166..8185
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
       61 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
      121 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ctggcactgt
      181 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
      241 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
      301 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
      361 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
      421 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
      481 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
      541 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
      601 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
      661 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
      721 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
      781 tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
      841 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
      901 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
      961 aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct tggccaccat
     1021 ggtgggccgc aacagcgcga ttgcagctgg agtgtgcggc gcgctgttca ttggctattg
     1081 catttacttt gatcgcaaac gccgcagcga tccgaacttt aaaggcggtg gagggtcctc
     1141 tagaggtggc tctggaggac tcgagaagag cctgagggcc accgtgagcg agagcagcat
     1201 gatcctgccc gagggcaagg gcagcttctg gatccactgg atcaccaccc aggacggctt
     1261 ctgcggcctg cccctggtga gcgtgaacga cggccacatc gtgggcatcc acggcctgac
     1321 cagcaacgac agcgagaaga acttcttcgt gcccctgacc gacggcttcg agaaggagta
     1381 cctggagaac gccgacaacc tgagctggga caagcactgg ttctgggagc ccagcaagat
     1441 cgcctggggc agcctgaacc tggtggagga gcagcccaag gaggagttca agatcagcaa
     1501 gctggtgagc gacctgttcg gcaacaccgt gaccgtgcag accgcatgcg gtggcggtgg
     1561 ctctggaggt ggtgggtccg gaggaggagg tggcggttcc ggcggaggtg ggtctggtgg
     1621 aggtggatct ggaggtggtg gatcaggagg aggaggttca ggcggccgcg agtttctggc
     1681 aaccacactg gaacggatcg agaaaaattt cgtgattact gatccgagac tgcctgacaa
     1741 cccaatcatt tttgcgagcg attccttcct gcagctgaca gaatattctc gggaagagat
     1801 cctggggcgc aattgccgtt ttctgcaggg acccgagaca gaccgtgcca ctgttcggaa
     1861 aatcagagat gctattgaca accagactga agtgaccgtt cagctgatca attataccaa
     1921 gagcggcaag aagttctgga acgtgttcca cctgcagccg atgcgcgatt ataagggcga
     1981 cgtccagtac ttcattggcg tgcagctgga tggcaccgaa cgtcttcatg gcgccgctga
     2041 gcgtgaggcg gtctgcctga tcaaaaagac agcctttcag attgctggat ccatgcttca
     2101 acttcctcct cttgaacgtc ttactctcat gttgcagcta ccaccattag agagactaac
     2161 actgagtaag agcgtgtaca agggcctgag ggactacagc ggcatcagca ccctgatctg
     2221 ccagctgacc aacagcagcg acggccacaa ggagaccatg ttcggcgtgg gctacggcag
     2281 cttcatcatc accaacggcc acctgttcag gaggaacaac ggcatgctga ccgtgaagac
     2341 ctggcacggc gagttcgtga tccacaacac cacccagctg aagatccact tcatccaggg
     2401 cagggacgtg atcctgatca ggatgcccaa ggacttcccc cccttcggca agaggaacct
     2461 gttcaggcag cccaagaggg aggagagggt gtgcatggtg ggcaccaact tccaggagga
     2521 attcctggca accacactgg aacggatcga gaaaaatttc gtgattactg atccgagact
     2581 gcctgacaac ccaatcattt ttgcgagcga ttccttcctg cagctgacag aatattctcg
     2641 ggaagagatc ctggggcgca attgccgttt tctgcaggga cccgagacag accgtgccac
     2701 tgttcggaaa atcagagatg ctattgacaa ccagactgaa gtgaccgttc agctgatcaa
     2761 ttataccaag agcggcaaga agttctggaa cgtgttccac ctgcagccga tgcgcgatta
     2821 taagggcgac gtccagtact tcattggcgt gcagctggat ggcaccgaac gtcttcatgg
     2881 cgccgctgag cgtgaggcgg tctgcctgat caaaaagaca gcctttcaga ttgctgagag
     2941 cgtgagcctg caaggtaccg gtatgggtga aaaacccggg accagggtct tcaagaagtc
     3001 gagccctaac tgcaagctca ccgtgtactt gggcaagcgt gactttgtgg atcacttgga
     3061 caaagtggat cctgtcgatg gtgtggtgct tgtggatcct gactacttga aggaccggaa
     3121 agtgtttgtg accctcacct gtgccttccg ctatggccga gaagacctgg atgtactggg
     3181 cctgtctttc cgcaaagatc tgttcatcgc cacctaccag gccttccccc ccatgcccaa
     3241 cccacctcgg ccccccaccc gcctacagga ccgactgctg aagaagttgg gccagcacgc
     3301 ccaccccttt tttttcacaa taccccagaa tttgccttgc tccgtcacac tgcagccagg
     3361 accggaggac acagggaagg cctgtggagt agactttgag attcgagcct tctgtgccaa
     3421 atctatagaa gaaaaaagcc acaaaaggaa ctccgtgcgg cttatcatca gaaaggtaca
     3481 gtttgctcct gagacacccg gcccccagcc atcagctgaa accacacgcc acttcctcat
     3541 gtctgaccgg aggtccctgc acctagaggc ttccctggac aaagagctgt actaccatgg
     3601 ggaacccctc aatgtcaacg tccacgtcac caacaattct gccaaaaccg tcaagaagat
     3661 cagagtgtct gtgagacagt atgccgacat ttgcctcttc agcaccgcgc agtacaagtg
     3721 tcctgtggct cagcttgaac aagatgacca ggtgtctccc agttccacat tctgcaaggt
     3781 gtacaccata accccgctgc tcagtgacaa ccgagagaag cgtggccttg cccttgatgg
     3841 gcaactcaag cacgaagaca ccaacctggc ttccagcacc attgtgaagg agggagccaa
     3901 caaggaggtg ctgggaatcc tagtatccta cagggtcaag gtgaagctgg tggtgtctcg
     3961 aggcggggat gtctccgtgg agctaccttt cgtcctaatg caccccaagc cccacgacca
     4021 catcaccctt ccccgacccc agtcagcccc ccgggaaata gacatccctg tggataccaa
     4081 cctcattgaa ttcgatacca actatgccac agacgacgac atcgtgtttg aggactttgc
     4141 gaggcttcgg ctgaagggga tgaaggatga cgactgtgat gaccagttct gcctgagctc
     4201 actcgaaggt ggcggtggct ctggaggtgg tgggtccgga ggaggcggca tcgagatgtc
     4261 cagcatggtg agcgatacta gctgtacctt cccatcatct gacggaatct tctggaagca
     4321 ctggattcag actaaggacg gccagtgtgg cagcccactg gtgagcacac gagacggatt
     4381 catcgtgggg attcacagcg cctccaactt tacaaacacc aataactatt tcacctcagt
     4441 gccaaagaac tttatggagc tgctgaccaa ccaggaggcc cagcagtggg tgagcgggtg
     4501 gcgcctgaac gccgattccg tgctgtgggg cgggcacaag gtgtttatgg catgcggaag
     4561 cggagctact aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc
     4621 tgaattcggg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     4681 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     4741 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     4801 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     4861 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     4921 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     4981 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     5041 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     5101 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     5161 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     5221 caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca
     5281 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     5341 caagtagtct agcctcccta tagtgagtcg tattacgtag atccagacat gataagatac
     5401 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     5461 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     5521 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaattc
     5581 gcgtccgcgg cgccaatgca ttgggcccgg tacccagctt ttgttccctt tagtgagggt
     5641 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5701 tcacaattcc acacaacata cgagccggga gcataaagtg taaagcctgg ggtgcctaat
     5761 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5821 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5881 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcgggagc
     5941 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     6001 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     6061 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     6121 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     6181 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     6241 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     6301 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     6361 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     6421 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     6481 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     6541 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     6601 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     6661 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     6721 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6781 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6841 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6901 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6961 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     7021 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7081 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     7141 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7201 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7261 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7321 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7381 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7441 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     7501 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     7561 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7621 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7681 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     7741 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7801 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     7861 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     7921 aaagaataga ccgagatagg gttgagtgtt gttccggttt ggaacaagag tccactatta
     7981 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     8041 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     8101 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     8161 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     8221 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     8281 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     8341 cagtcgacc
//
