LOCUS       sequence-469012-        5285 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of uTEV3 tagged with an eGFP marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5285)
  AUTHORS   Silva-Rojas R, Vicente N, Gavilan-Herrera M, Labrador-Cantarero V, 
            Sicilia J, Gimenez-Saez O, Dumitru AC, Sanchez MI, Gato-Vilaseca M, 
            Velazquez-Carreras D, Lopez JA, Vazquez J, Herrero-Galan E, 
            Lopez-Unzu MA, Pricolo MR, Alegre-Cebollada J
  TITLE     Mechanically knocking out titin reveals protein tension loss as a 
            trigger of muscle disease.
  JOURNAL   Nat Biomed Eng. 2025 Jun 5. doi: 10.1038/s41551-025-01403-x.
  PUBMED    40473933
REFERENCE   2  (bases 1 to 5285)
  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"; journalName: "Nat Biomed 
            Eng. 2025 Jun 5. doi: 10.1038/s41551-025-01403-x."
COMMENT     Sequence Label: pCMV-eGFP-uTEV3
FEATURES             Location/Qualifiers
     source          1..5285
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        201..504
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        505..708
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     659..679
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     753..772
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        753..771
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      785..794
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(791..793,794..796,797..1507)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(791..793,794..796,797..1507)
                     /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(836..857)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1097..1116)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1444..1465
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1544..2251
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGTFWKHWIQTKDGQCGNPLVSTRDGFI
                     VGIHSASNFTNTNNYFASVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     primer_bind     complement(2306..2323)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2312..2519
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2548..2688
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2548..2677
                     /label=AAV2 ITR
     rep_origin      2763..3218
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2850..2869)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3060..3081
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3235..3254)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3354..3376
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3414..3432)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3500..3604
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3605..3673,3674..4465)
                     /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     complement(3823..3842)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4636..5224
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5125..5144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgttgtaca tagtaatcaa ttacggggtc attagttcat
      181 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      241 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      301 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      361 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      421 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      481 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      541 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      601 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      661 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      721 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      781 gcttgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
      841 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
      901 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
      961 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1021 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1081 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1141 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1201 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     1261 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     1321 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1381 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     1441 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     1501 gtacaaggac cttggatctg gcacaggcgg atccggttca ggcggagaat ccttgtttaa
     1561 gggaccacgt gattacaacc cgatatcgag caccatttgt catttgacga atgaatctga
     1621 tgggcacaca acatcgttgt atggtattgg atttggtccc ttcatcatta caaacaagca
     1681 cttgtttaga agaaataatg gaacactgtt ggtccaatca ctacatggtg tattcaaggt
     1741 caagaacacc acgactttgc aacaacacct cattgatggg agggacatga taattattcg
     1801 catgcctaag gatttcccac catttcctca aaagctgaaa tttagagagc cacaaaggga
     1861 agagcgcata tgtcttgtga caaccaactt ccaaactaag agcatgtcta gcatggtgtc
     1921 agacactagt tgcacattcc cttcatctga tggcacattc tggaagcatt ggattcaaac
     1981 caaggatggg cagtgtggca atccattagt atcaactaga gatgggttca ttgttggtat
     2041 acactcagca tcgaatttca ccaacacaaa caattatttc gcaagcgtgc cgaaaaactt
     2101 catggaattg ttgacaaatc aggaggcgca gcagtgggtt agtggttggc gattaaatgc
     2161 tgactcagta ttgtgggggg gccataaagt tttcatggtg aaacctgaag agccttttca
     2221 gccagttaag gaagcgactc aactcatgaa tgaattggtc tacagccagt agtaagaatt
     2281 cctcgagcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     2341 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     2401 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2461 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggag
     2521 gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct
     2581 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     2641 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     2701 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     2761 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2821 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2881 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2941 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     3001 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3061 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     3121 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     3181 taacgcgaat tttaacaaaa tattaacgtt tacaattttg tggtgcactc tcagtacaat
     3241 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     3301 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3361 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     3421 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     3481 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3541 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3601 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3661 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3721 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     3781 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     3841 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     3901 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     3961 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     4021 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     4081 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     4141 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     4201 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     4261 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     4321 aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     4381 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     4441 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     4501 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4561 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4621 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     4681 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     4741 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     4801 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     4861 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     4921 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     4981 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5041 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5101 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5161 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5221 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5281 catgt
//
