LOCUS       sequence-267522-        5747 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of uFLARE protease component.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5747)
  AUTHORS   Sanchez MI, Ting AY
  TITLE     Directed evolution improves the catalytic efficiency of TEV 
            protease.
  JOURNAL   Nat Methods. 2019 Dec 9. pii: 10.1038/s41592-019-0665-7. doi: 
            10.1038/s41592-019-0665-7.
  PUBMED    31819267
REFERENCE   2  (bases 1 to 5747)
  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 
            Methods. 2019 Dec 9. pii: 10.1038/s41592-019-0665-7. doi: 
            10.1038/s41592-019-0665-7."
COMMENT     Sequence Label: CMV:eGFP-CaM-uTEV1-Delta(220-242)
FEATURES             Location/Qualifiers
     source          1..5747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        204..407
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     358..378
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     452..471
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        452..470
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      484..493
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(490..492,493..495,496..1206)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(490..492,493..495,496..1206)
                     /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(535..556)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(796..815)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1143..1164
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1243..1686
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="MDQLTEEQIAEFKEAFSLLDKDGDGTITTKELGTGMRSLGQNPTE
                     AELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGNGYISAAE
                     LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             1687..1728
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2467..2484)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2473..2680
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2709..2849
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2709..2838
                     /label=AAV2 ITR
     rep_origin      2924..3379
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3011..3030)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3221..3242
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3396..3415)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3515..3537
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3575..3593)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3661..3765
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3766..3834,3835..4626)
                     /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(3984..4003)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4797..5385
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5286..5305
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5447..5576
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        join(5647..5747,1..203)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
       61 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      121 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      181 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      241 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      301 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      361 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
      421 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
      481 cttgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
      541 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
      601 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
      661 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
      721 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
      781 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
      841 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
      901 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
      961 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     1021 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1081 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     1141 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     1201 tacaaggacc ttggatctgg cacaggcgga tccggttcag gcatggacca actgactgaa
     1261 gagcagatcg cagaatttaa agaggctttc tccctactgg acaaggacgg ggatgggaca
     1321 ataacaacca aggagctggg gacggggatg cggtctctgg ggcagaaccc cacagaagca
     1381 gagctgcagg acatgatcaa tgaagtagat gccgacggtg acggcacaat cgacttccct
     1441 gagttcctga caatgatggc aagaaaaatg aaatacacag acagtgaaga agaaattaga
     1501 gaagcgttcc gtgtgtttga taaggatggc aatggctaca tcagtgcagc agagcttcgc
     1561 cacgtgatga caaaccttgg agagaagtta acagatgaag aggttgatga aatgatcagg
     1621 gaagcagaca tcgatgggga tggtcaggta aactacgaag agtttgtaca aatgatgaca
     1681 gcgaagggca agccgatccc taaccctctg ctgggcctgg acagcactgg tggaagtgga
     1741 tcaggcagcg gtggatctgg gggtggcggt accgggggag aatccttgtt taagggacca
     1801 cgtgattaca acccgatatc gagcaccatt tgtcatttga cgaatgaatc tgatgggcac
     1861 acaacatcgt tgtatggtat tggatttggt cccttcatca ttacaaacaa gcacttgttt
     1921 agaagaaata atggaacact gttggtccaa tcactacatg gtgtattcaa ggtcaagaac
     1981 accacgactt tgcaacaaca cctcattgat gggagggaca tgataattat tcgcatgcct
     2041 aaggatttcc caccatttcc tcaaaagctg aaatttagag agccacaaag ggaagagcgc
     2101 atatgtcttg tgacaaccaa cttccaaact aagagcatgt ctagcatggt gtcagacact
     2161 agttgcacat tcccttcatc tgatggcata ttctggaagc attggattca aaccaaggat
     2221 gggcagtgtg gcaatccatt agtatcaact agagatgggt tcattgttgg tatacactca
     2281 gcatcgaatt tcaccaacac aaacaattat ttcacaagcg tgccgaaaaa cttcatggaa
     2341 ttgttgacaa atcaggaggc gcagcagtgg gttagtggtt ggcgattaaa tgctgactca
     2401 gtattgtggg ggggccataa agttttcatg gtgtaagaat tcctcgagct agagctcgct
     2461 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2521 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2581 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     2641 agggggagga ttgggaagac aatagcaggc atgctgggga ggtaaccacg tgcggaccga
     2701 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     2761 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     2821 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     2881 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     2941 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3001 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3061 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3121 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3181 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3241 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     3301 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3361 atattaacgt ttacaatttt gtggtgcact ctcagtacaa tctgctctga tgccgcatag
     3421 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3481 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3541 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     3601 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     3661 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3721 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     3781 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     3841 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3901 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3961 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     4021 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4081 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     4141 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     4201 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     4261 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     4321 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     4381 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     4441 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     4501 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     4561 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     4621 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4681 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4741 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4801 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4861 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4921 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4981 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5041 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5101 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5161 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5221 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5281 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5341 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5401 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg
     5461 cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc
     5521 agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg
     5581 ccgcacgcgt tgtacatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     5641 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     5701 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaatag
//
