LOCUS       sequence-261626-        5746 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 5746)
  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 5746)
  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..5746
                     /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..1987
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="MDQLTEEQIAEFKEAFSLLDKDGDGTITTKELGTGMRSLGQNPTE
                     AELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGNGYISAAE
                     LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             1988..2029
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2768..2785)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2774..2981
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3010..3150
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3010..3139
                     /label=AAV2 ITR
     rep_origin      3224..3679
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3311..3330)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3521..3542
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3696..3715)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3815..3837
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3875..3893)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3961..4065
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4066..4134,4135..4926)
                     /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(4284..4303)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5097..5685
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5586..5605
                     /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 atgggtggac 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 ggcatggacc aactgactga
     1561 agagcagatc gcagaattta aagaggcttt ctccctactg gacaaggacg gggatgggac
     1621 aataacaacc aaggagctgg ggacggggat gcggtctctg gggcagaacc ccacagaagc
     1681 agagctgcag gacatgatca atgaagtaga tgccgacggt gacggcacaa tcgacttccc
     1741 tgagttcctg acaatgatgg caagaaaaat gaaatacaca gacagtgaag aagaaattag
     1801 agaagcgttc cgtgtgtttg ataaggatgg caatggctac atcagtgcag cagagcttcg
     1861 ccacgtgatg acaaaccttg gagagaagtt aacagatgaa gaggttgatg aaatgatcag
     1921 ggaagcagac atcgatgggg atggtcaggt aaactacgaa gagtttgtac aaatgatgac
     1981 agcgaagggc aagccgatcc ctaaccctct gctgggcctg gacagcactg gtggaagtgg
     2041 atcaggcagc ggtggatctg ggggtggcgg taccggggga gaatccttgt ttaagggacc
     2101 acgtgattac aacccgatat cgagcaccat ttgtcatttg acgaatgaat ctgatgggca
     2161 cacaacatcg ttgtatggta ttggatttgg tcccttcatc attacaaaca agcacttgtt
     2221 tagaagaaat aatggaacac tgttggtcca atcactacat ggtgtattca aggtcaagaa
     2281 caccacgact ttgcaacaac acctcattga tgggagggac atgataatta ttcgcatgcc
     2341 taaggatttc ccaccatttc ctcaaaagct gaaatttaga gagccacaaa gggaagagcg
     2401 catatgtctt gtgacaacca acttccaaac taagagcatg tctagcatgg tgtcagacac
     2461 tagttgcaca ttcccttcat ctgatggcat attctggaag cattggattc aaaccaagga
     2521 tgggcagtgt ggcaatccat tagtatcaac tagagatggg ttcattgttg gtatacactc
     2581 agcatcgaat ttcaccaaca caaacaatta tttcacaagc gtgccgaaaa acttcatgga
     2641 attgttgaca aatcaggagg cgcagcagtg ggttagtggt tggcgattaa atgctgactc
     2701 agtattgtgg gggggccata aagttttcat ggtgtaagaa ttcctcgagc tagagctcgc
     2761 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2821 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2881 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2941 aagggggagg attgggaaga caatagcagg catgctgggg aggtaaccac gtgcggaccg
     3001 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     3061 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     3121 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     3181 atctgtgcgg tattcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     3241 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3301 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3361 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3421 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3481 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3541 ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     3601 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3661 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3721 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3781 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3841 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     3901 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     3961 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     4021 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     4081 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4141 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     4201 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     4261 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     4321 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4381 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     4441 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     4501 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     4561 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     4621 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     4681 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     4741 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     4801 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     4861 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     4921 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4981 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5041 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5101 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5161 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5221 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     5281 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5341 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5401 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5461 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5521 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5581 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5641 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5701 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
