LOCUS       sequence-267454-        6659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of uFLARE TF component.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6659)
  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 6659)
  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:CD4-HA-CIBN-MKII-NNES-hLOV-TEVcs(ENLYFQ/M)...
FEATURES             Location/Qualifiers
     source          1..6659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     553..572
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        553..571
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      585..594
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     591..650
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             675..701
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             702..728
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1476..1985
                     /codon_start=1
                     /product="N-terminal portion of the CIB1 transcription 
                     factor from Arabidopsis thaliana"
                     /label=CIBN
                     /note="binds to CRY2 (cryptochrome 2) that has absorbed 
                     blue light (Kennedy et al., 2010)"
                     /translation="MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSS
                     MITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKAAKFDTETKDCN
                     EAAKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKE
                     LEKTDYI"
     CDS             2607..2627
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQM"
     CDS             2640..2663
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2664..3101
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="KLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKR
                     SPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKD
                     AVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             3102..3446
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="activation domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 activation domain
                     /translation="ANFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSN
                     VHDNFMNNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDD
                     EDTPPNPKKE"
     primer_bind     complement(3480..3497)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3486..3693
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3722..3862
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3722..3851
                     /label=AAV2 ITR
     rep_origin      3937..4392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4024..4043)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4234..4255
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4409..4428)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4528..4550
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4588..4606)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4674..4778
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4779..4847,4848..5639)
                     /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(4997..5016)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5810..6398
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6299..6318
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6460..6589
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
      541 gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgccacc atggagacag
      601 acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt gacggggccc
      661 agccggccag atcttaccca tacgatgtgc cagattacgc ctatccctat gacgtccctg
      721 attacgctct cgacttccag aaggcctcca gcatagtcta taagaaagag ggggaacagg
      781 tggagttctc cttcccactc gcctttacag ttgaaaagct gacgggcagt ggcgagctgt
      841 ggtggcaggc ggagagggct tcctcctcca agtcttggat cacctttgac ctgaagaaca
      901 aggaagtgtc tgtaaaacgg gttacccagg accctaagct ccagatgggc aagaagctcc
      961 cgctccacct caccctgccc caggccttgc ctcagtatgc tggctctgga aacctcaccc
     1021 tggcccttga agcgaaaaca ggaaagttgc atcaggaagt gaacctggtg gtgatgagag
     1081 ccactcagct ccagaaaaat ttgacctgtg aggtgtgggg acccacctcc cctaagctga
     1141 tgctgagctt gaaactggag aacaaggagg caaaggtctc gaagcgggag aaggcggtgt
     1201 gggtgctgaa ccctgaggcg gggatgtggc agtgtctgct gagtgactcg ggacaggtcc
     1261 tgctggaatc caacatcaag gttctgccca catggtccac cccggtgcag ccaatggccc
     1321 tgattgtgct ggggggcgtc gccggcctcc tgcttttcat tgggctaggc atcttcttct
     1381 gtgtcaggtg ccggcaccga aggcgcaagg gctcgggctc gacctcgggc tcgggcagcg
     1441 gtggctctag aggttctggt ggcagctctg gaggtatgaa tggagctata ggaggtgacc
     1501 ttttgctcaa ttttcctgac atgtcggtcc tagagcgcca aagggctcac ctcaagtacc
     1561 tcaatcccac ctttgattct cctctcgccg gcttctttgc cgattcttca atgattaccg
     1621 gcggcgagat ggacagctat ctttcgactg ccggtttgaa tcttccgatg atgtacggtg
     1681 agacgacggt ggaaggtgat tcaagactct caatttcgcc ggaaacgacg cttgggactg
     1741 gaaatttcaa ggcagcgaag tttgatacag agactaagga ttgtaatgag gcggcgaaga
     1801 agatgacgat gaacagagat gacctagtag aagaaggaga agaagagaag tcgaaaataa
     1861 cagagcaaaa caatgggagc acaaaaagca tcaagaagat gaaacacaaa gccaagaaag
     1921 aagagaacaa tttctctaat gattcatcta aagtgacgaa ggaattggag aaaacggatt
     1981 atattcatgg tggctctggt agcttcaacg ctcgcaggaa gctggctggt gctatcctgt
     2041 tcaccatgct cgccacaaga aacttcagcg gcagctttaa tgcgcggcgc aagttggccg
     2101 gagccatact tttcacaatg ttggctacca gaaatttttc ggagctggcc gagaagctgg
     2161 ccggcctgga catcaacggt ggagctagcg agtttcgggc aaccacactg gaacggatcg
     2221 agaaaagctt cgtgattact gatccgagac tgcctgacaa cccaatcatt tttgtgagcg
     2281 attccttcct gcagctgaca gaatattctc gggaagagat cctggggcgc aattgccgtt
     2341 ttctgcaggg acccgagaca gaccgtgcca ctgttcggaa aatcagagat gctattgaca
     2401 accagactga agtgaccgtt cagctgatca attataccaa gagcggcaag aagttctgga
     2461 acgtgttcca cctgcagccg atgcgcgatt ataagggcga cgtccagtac ttcattggcg
     2521 tgcagctgga tggcaccgaa cgtcttcatg gcgccgctga gcgtgaggcg gtctgcctgg
     2581 tcaaaaagac agcctttcag attgctgaga acctgtactt ccagatgggc ggaggatccg
     2641 actacaagga tgacgacgat aagaagctac tgtcttctat cgaacaagca tgcgatattt
     2701 gccgacttaa aaagctcaag tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga
     2761 acaactggga gtgtcgctac tctcccaaaa ccaaaaggtc tccgctgact agggcacatc
     2821 tgacagaagt ggaatcaagg ctagaaagac tggaacagct atttctactg atttttcctc
     2881 gagaagacct tgacatgatt ttgaaaatgg attctttaca ggatataaaa gcattgttaa
     2941 caggattatt tgtacaagat aatgtgaata aagatgccgt cacagataga ttggcttcag
     3001 tggagactga tatgcctcta acattgagac agcatagaat aagtgcgaca tcatcatcgg
     3061 aagagagtag taacaaaggt caaagacagt tgactgtatc ggccaatttt aatcaaagtg
     3121 ggaatattgc tgatagctca ttgtccttca ctttcactaa cagtagcaac ggtccgaacc
     3181 tcataacaac tcaaacaaat tctcaagcgc tttcacaacc aattgcctcc tctaacgttc
     3241 atgataactt catgaataat gaaatcacgg ctagtaaaat tgatgatggt aataattcaa
     3301 aaccactgtc acctggttgg acggaccaaa ctgcgtataa cgcgtttgga atcactacag
     3361 ggatgtttaa taccactaca atggatgatg tatataacta tctattcgat gatgaagata
     3421 ccccaccaaa cccaaaaaaa gagtagtaag aattcctcga gctagagctc gctgatcagc
     3481 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     3541 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     3601 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     3661 ggattgggaa gacaatagca ggcatgctgg ggaggtaacc acgtgcggac cgagcggccg
     3721 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     3781 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     3841 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     3901 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     3961 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     4021 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4081 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4141 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4201 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4261 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     4321 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4381 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4441 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4501 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     4561 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     4621 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4681 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4741 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     4801 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     4861 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     4921 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     4981 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5041 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5101 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5161 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5221 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5281 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5341 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5401 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     5461 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     5521 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     5581 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     5641 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     5701 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     5761 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     5821 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     5881 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     5941 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6001 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6061 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6121 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6181 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6241 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6301 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6361 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6421 tacggttcct ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc
     6481 gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc
     6541 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcacg
     6601 cgttgtacat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccg
//
