LOCUS       sequence-153654-        5944 bp    DNA     circular SYN 14-APR-2025
DEFINITION  mammalian expression of Zdk1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5944)
  AUTHORS   Wang H, Vilela M, Winkler A, Tarnawski M, Schlichting I, Yumerefendi
            H, Kuhlman B, Liu R, Danuser G, Hahn KM
  TITLE     LOVTRAP: an optogenetic system for photoinduced protein 
            dissociation.
  JOURNAL   Nat Methods. 2016 Jul 18. doi: 10.1038/nmeth.3926.
  PUBMED    27427858
REFERENCE   2  (bases 1 to 5944)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2016 Jul 18. doi: 10.1038/nmeth.3926."
COMMENT     Sequence Label: pTriEx-mVenus-Zdk1
FEATURES             Location/Qualifiers
     source          1..5944
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     110..937
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             110..234
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(272..877)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1005..1308
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1310..1512
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1463..1483
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1715..1737
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1799..1818
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1860..1879
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1860..1878
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1879..1903
                     /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        1919..2028
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             join(2043..2045,2046..2048,2049..2759)
                     /codon_start=1
                     /product="photostable derivative of mVenus (Lee et al., 
                     2020)"
                     /label=mGold
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFISTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2088..2109)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2349..2368)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2696..2717
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2778..2954
                     /codon_start=1
                     /product="""Zdark"" protein, derived from the Z subunit of 
                     protein A, that binds to the dark state of LOV2 and is 
                     released upon irradiation (Wang et al., 2016)"
                     /label=Zdk1
                     /translation="MVDNKFNKEKTRAGAEIHSLPNLNVEQKFAFIVSLFDDPSQSANL
                     LAEAKKLNDAQAPK"
     CDS             2973..2990
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2997..3017
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3024..3047
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3087..3119
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             3126..3149
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3239..3258)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3304..3359
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3358..3377)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3447..3494
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3507..4212
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3595..3967
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3624..4212)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4221..4254)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(4422..5009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4501..4520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5022..5813,5814..5882))
                     /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     5645..5664
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aactgttaca ttgcaaacgt ggtttcgtgt gccaagtgtg aaaaccgatg tttaatcaag
       61 gctctgacgc atttctacaa ccacgactcc aagtgtgtgg gtgaagtcat gcatctttta
      121 atcaaatccc aagatgtgta taaaccacca aactgccaaa aaatgaaaac tgtcgacaag
      181 ctctgtccgt ttgctggcaa ctgcaagggt ctcaatccta tttgtaatta ttgaataata
      241 aaacaattat aaatgtcaaa tttgtttttt attaacgata caaaccaaac gcaacaagaa
      301 catttgtagt attatctata attgaaaacg cgtagttata atcgctgagg taatatttaa
      361 aatcattttc aaatgattca cagttaattt gcgacaatat aattttattt tcacataaac
      421 tagacgcctt gtcgtcttct tcttcgtatt ccttctcttt ttcatttttc tcttcataaa
      481 aattaacata gttattatcg tatccatata tgtatctatc gtatagagta aattttttgt
      541 tgtcataaat atatatgtct tttttaatgg ggtgtatagt accgctgcgc atagtttttc
      601 tgtaatttac aacagtgcta ttttctggta gttcttcgga gtgtgttgct ttaattatta
      661 aatttatata atcaatgaat ttgggatcgt cggttttgta caatatgttg ccggcatagt
      721 acgcagcttc ttctagttca attacaccat tttttagcag caccggatta acataacttt
      781 ccaaaatgtt gtacgaaccg ttaaacaaaa acagttcacc tcccttttct atactattgt
      841 ctgcgagcag ttgtttgttg ttaaaaataa cagccattgt aatgagacgc acaaactaat
      901 atcacaaact ggaaatgtct atcaatatat agttgctgat ggccggccta ttaatagtaa
      961 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     1021 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     1081 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     1141 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt
     1201 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac
     1261 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgct gatgcggttt
     1321 tggcagtaca ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     1381 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     1441 cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     1501 ataagcagac gtcgtttagt gaaccgtcag atcactagat gctttattgc ggtagtttat
     1561 cacagttaaa ttgctaacgc cagtctcgaa cttaacgtgc agaagttggt cgtgaggcac
     1621 tgggcaggta agtatcgggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg
     1681 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     1741 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     1801 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcg gaccgaaatt
     1861 aatacgactc actatagggg aattgtgagc ggataacaat tccccggagt taatccggga
     1921 cctttaattc aacccaacac aatatattat agttaaataa gaattattat caaatcattt
     1981 gtatattaat taaaatacta tactgtaaat tacattttat ttacaatcaa aggagatata
     2041 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     2101 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     2161 acggcaagct gaccctgaag ttcatcagca ccaccggcaa gctgcccgtg ccctggccca
     2221 ccctcgtgac caccctcggc tacggcctgc agtgcttcgc ccgctacccc gaccacatga
     2281 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     2341 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2401 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2461 acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga
     2521 acggcatcaa ggcaaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg
     2581 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     2641 actacctgag ctaccagtcc aaactgagca aagaccccaa cgagaagcgc gatcacatgg
     2701 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     2761 gttctggaag tggatccatg gtggataaca aattcaataa agaaaagacg cgtgccggtg
     2821 cggaaatcca ttctctgcca aacctgaatg ttgagcagaa gtttgccttc atcgtgagcc
     2881 tgtttgatga tccatctcag agcgccaatc tgctggccga agccaaaaaa ctgaacgatg
     2941 cccaggcccc aaaaggtggt tctggtggta gccatcatca tcatcatcat ggcggtgaaa
     3001 atctgtattt ccagggcggc cgcgactaca aggacgacga cgacaaataa aagcttgcgg
     3061 ccgcacagct gtatacacgt gcaagccagc cagaactcgc cccggaagac cccgaggatc
     3121 tcgagcacca ccatcaccat caccatcact aagtgattaa cctcaggtgc aggctgccta
     3181 tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact gagatcgatc
     3241 tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg
     3301 gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac
     3361 tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga
     3421 gtttggcaac atatgcccat atgtaactag cataacccct tggggcctct aaacgggtct
     3481 tgaggggttt tttgctgaaa gcatgcggag gaaattctcc ttgaagtttc cctggtgttc
     3541 aaagtaaagg agtttgcacc agacgcacct ctgttcactg gtccggcgta ttaaaacacg
     3601 atacattgtt attagtacat ttattaagcg ctagattctg tgcgttgttg atttacagac
     3661 aattgttgta cgtattttaa taattcatta aatttataat ctttagggtg gtatgttaga
     3721 gcgaaaatca aatgattttc agcgtcttta tatctgaatt taaatattaa atcctcaata
     3781 gatttgtaaa ataggtttcg attagtttca aacaagggtt gtttttccga accgatggct
     3841 ggactatcta atggattttc gctcaacgcc acaaaacttg ccaaatcttg tagcagcaat
     3901 ctagctttgt cgatattcgt ttgtgttttg ttttgtaata aaggttcgac gtcgttcaaa
     3961 atattatgcg cttttgtatt tctttcatca ctgtcgttag tgtacaattg actcgacgta
     4021 aacacgttaa atagagcttg gacatattta acatcgggcg tgttagcttt attaggccga
     4081 ttatcgtcgt cgtcccaacc ctcgtcgtta gaagttgctt ccgaagacga ttttgccata
     4141 gccacacgac gcctattaat tgtgtcggct aacacgtccg cgatcaaatt tgtagttgag
     4201 ctttttggaa ttgcgatcgc ataacttcgt atagcataca ttatacgaag ttataagctc
     4261 ggaacgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4321 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4381 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4441 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     4501 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     4561 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct
     4621 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     4681 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4741 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4801 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4861 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4921 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4981 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gttaccaatg cttaatcagt
     5041 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5101 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5161 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5221 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5281 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5341 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5401 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5461 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5521 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5581 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5641 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5701 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5761 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5821 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5881 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgtccgcg
     5941 cgtt
//
