LOCUS       sequence-324824-        7852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTriEx based vector for protein expression in mammalian cells for 
            targets fused with C-terminal uv-GFP, Flag and His.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7852)
  AUTHORS   Bruni R, Kloss B
  TITLE     High-throughput cloning and expression of integral membrane proteins
            in Escherichia coli.
  JOURNAL   Curr Protoc Protein Sci. 2013 Nov 5;74:29.6.1-29.6.34. doi: 
            10.1002/0471140864.ps2906s74.
  PUBMED    24510647
REFERENCE   2  (bases 1 to 7852)
  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: "Curr Protoc
            Protein Sci."; date: "2013-11-5"; volume: "74:29.6.1-29.6.34. doi"; 
            pages: " 10.1002/0471140864.ps2906s74"
COMMENT     Sequence Label: pRB30-GFP
FEATURES             Location/Qualifiers
     source          1..7852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1140..1443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1449..1726
                     /label=chicken beta-actin promoter
     primer_bind     2005..2027
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2089..2108
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     2150..2169
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2150..2168
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2169..2193
                     /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        2209..2318
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        2365..2797
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(2798..2884,2885..4219)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     CDS             4278..4298
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4299..5009
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(4326..4354)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4949..4969
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             5010..5039
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             5040..5063
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(5147..5166)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5212..5267
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5266..5285)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      5355..5402
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     5415..6120
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           5503..5875
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(5532..6120)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(6129..6162)
                     /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(6330..6917)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6409..6428)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6930..7721,7722..7790))
                     /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     7553..7572
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggccg
     1021 taatgagacg cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctct
     1081 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     1141 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     1201 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     1261 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     1321 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     1381 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     1441 atgcatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     1501 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     1561 gggggggggg cgcgcgccrg gsggggsggg gsggggsgrg gggsggggsg gggsgrggcg
     1621 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
     1681 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
     1741 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
     1801 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctccttcgg gctgtaatta
     1861 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
     1921 ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc
     1981 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
     2041 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     2101 ttattgtgct gtctcatcat tttggcaaag aattggatcg gaccgaaatt aatacgactc
     2161 actatagggg aattgtgagc ggataacaat tccccggagt taatccggga cctttaattc
     2221 aacccaacac aatatattat agttaaataa gaattattat caaatcattt gtatattaat
     2281 taaaatacta tactgtaaat tacattttat ttacaatcaa aggagatata ccctggcctt
     2341 aagaaggaga tatactatgc aggtcgttca ctattattta gtgaaatgag atattatgat
     2401 attttctgaa ttgtgattaa aaaggcaact ttatgcccat gcaacagaaa ctataaaaaa
     2461 tacagagaat gaaaagaaac agatagattt tttagttctt taggcccgta gtctgcaaat
     2521 ccttttatga ttttctatca aacaaaagag gaaaatagac cagttgcaat ccaaacgaga
     2581 gtctaataga atgaggtcga aaagtaaatc gcgcgggttt gttactgata aagcaggcaa
     2641 gacctaaaat gtgtaaaggg caaagtgtat actttggcgt caccccttac atattttagg
     2701 tcttttttta ttgtgcgtaa ctaacttgcc atcttcaaac aggagggctg gaagaagcag
     2761 accgctaaca cagtacataa aaaaggagac atgaacgatg aacatcaaaa agtttgcaaa
     2821 acaagcaaca gtattaacct ttactaccgc actgctggca ggaggcgcaa ctcaagcgtt
     2881 tgcgaaagaa acgaaccaaa agccatataa ggaaacatac ggcatttccc atattacacg
     2941 ccatgatatg ctgcaaatcc ctgaacagca aaaaaatgaa aaatataaag ttcctgagtt
     3001 cgattcgtcc acaattaaaa atatctcttc tgcaaaaggc ctggacgttt gggacagctg
     3061 gccattacaa aacactgacg gcactgtcgc aaactatcac ggctaccaca tcgtctttgc
     3121 attagccgga gatcctaaaa atgcggatga cacatcgatt tacatgttct atcaaaaagt
     3181 cggcgaaact tctattgaca gctggaaaaa cgctggccgc gtctttaaag acagcgacaa
     3241 attcgatgca aatgattcta tcctaaaaga ccaaacacaa gaatggtcag gttcagccac
     3301 atttacatct gacggaaaaa tccgtttatt ctacactgat ttctccggta aacattacgg
     3361 caaacaaaca ctgacaactg cacaagttaa cgtatcagca tcagacagct ctttgaacat
     3421 caacggtgta gaggattata aatcaatctt tgacggtgac ggaaaaacgt atcaaaatgt
     3481 acagcagttc atcgatgaag gcaactacag ctcaggcgac aaccatacgc tgagagatcc
     3541 tcactacgta gaagataaag gccacaaata cttagtattt gaagcaaaca ctggaactga
     3601 agatggctac caaggcgaag aatctttatt taacaaagca tactatggca aaagcacatc
     3661 attcttccgt caagaaagtc aaaaacttct gcaaagcgat aaaaaacgca cggctgagtt
     3721 agcaaacggc gctctcggta tgattgagct aaacgatgat tacacactga aaaaagtgat
     3781 gaaaccgctg attgcatcta acacagtaac agatgaaatt gaacgcgcga acgtctttaa
     3841 aatgaacggc aaatggtacc tgttcactga ctcccgcgga tcaaaaatga cgattgacgg
     3901 cattacgtct aacgatattt acatgcttgg ttatgtttct aattctttaa ctggcccata
     3961 caagccgctg aacaaaactg gccttgtgtt aaaaatggat cttgatccta acgatgtaac
     4021 ctttacttac tcacacttcg ctgtacctca agcgaaagga aacaatgtcg tgattacaag
     4081 ctatatgaca aacagaggat tctacgcaga caaacaatca acgtttgcgc ctagcttcct
     4141 gctgaacatc aaaggcaaga aaacatctgt tgtcaaagac agcatccttg aacaaggaca
     4201 attaacagtt aacaaataaa aacgcaaaag aaaatgccga tatcctattg gcattgacgt
     4261 caggtggcac acctgcagag aacctctact tccaatcgag caaaggagaa gaacttttca
     4321 ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg
     4381 tcagtggaga gggtgaaggt gatgctacat acggaaagct tacccttaaa tttatttgca
     4441 ctactggaaa actacctgtt ccctggccaa cacttgtcac tactttctct tatggtgttc
     4501 aatgcttttc ccgttatccg gatcatatga aacggcatga ctttttcaag agtgccatgc
     4561 ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga tgacgggaac tacaagacgc
     4621 gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg tatcgagtta aaaggtattg
     4681 attttaaaga agatggaaac attctcggac acaaacttga gtacaactat aactcacaca
     4741 atgtatacat cacggcagac aaacaaaaga atggaatcaa agctaacttc aaaattcgcc
     4801 acaacattga agatggatcc gttcaactag cagaccatta tcaacaaaat actccaattg
     4861 gcgatggccc tgtcctttta ccagacaacc attacctgtc gacacaatct gccctttcga
     4921 aagatcccaa cgaaaagcgt gaccacatgg tccttcttga gtttgtaact gctgctggga
     4981 ttacacatgg catggatgag ctctacaaac accatcatca ccatcaccat caccaccatg
     5041 attacaagga tgacgacgat aagtgaggcc tcaggtgcag gctgcctatc agaaggtggt
     5101 ggctggtgtg gccaatgccc tggctcacaa ataccactga gatcgatctt tttccctctg
     5161 ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga
     5221 aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat
     5281 atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat
     5341 atgcccatat gtaactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     5401 tgctgaaagc atgcggagga aattctcctt gaagtttccc tggtgttcaa agtaaaggag
     5461 tttgcaccag acgcacctct gttcactggt ccggcgtatt aaaacacgat acattgttat
     5521 tagtacattt attaagcgct agattctgtg cgttgttgat ttacagacaa ttgttgtacg
     5581 tattttaata attcattaaa tttataatct ttagggtggt atgttagagc gaaaatcaaa
     5641 tgattttcag cgtctttata tctgaattta aatattaaat cctcaataga tttgtaaaat
     5701 aggtttcgat tagtttcaaa caagggttgt ttttccgaac cgatggctgg actatctaat
     5761 ggattttcgc tcaacgccac aaaacttgcc aaatcttgta gcagcaatct agctttgtcg
     5821 atattcgttt gtgttttgtt ttgtaataaa ggttcgacgt cgttcaaaat attatgcgct
     5881 tttgtatttc tttcatcact gtcgttagtg tacaattgac tcgacgtaaa cacgttaaat
     5941 agagcttgga catatttaac atcgggcgtg ttagctttat taggccgatt atcgtcgtcg
     6001 tcccaaccct cgtcgttaga agttgcttcc gaagacgatt ttgccatagc cacacgacgc
     6061 ctattaattg tgtcggctaa cacgtccgcg atcaaatttg tagttgagct ttttggaatt
     6121 gcgatcgcat aacttcgtat agcatacatt atacgaagtt ataagctcgg aacgctgcgc
     6181 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     6241 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     6301 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgccccct gacgagcatc
     6361 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6421 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6481 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     6541 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6601 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6661 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6721 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6781 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6841 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6901 gaaaaaaagg atctcaagaa gatcctttgt taccaatgct taatcagtga ggcacctatc
     6961 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     7021 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     7081 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7141 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7201 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7261 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7321 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7381 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7441 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7501 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7561 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7621 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7681 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7741 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7801 tttcaatatt attgaagcat ttatcagggt tattgtctca tgtccgcgcg tt
//
