LOCUS       sequence-324826-        7237 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTriEx based vector for protein expression in mammalian cells for 
            targets fused with N-terminal Flag and His.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7237)
  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 7237)
  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: pRB33
FEATURES             Location/Qualifiers
     source          1..7237
                     /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"
     CDS             2339..2368
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             2372..2395
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2399..2419
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     protein_bind    complement(2449..2482)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2497..2942
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(2943..3029,3030..4364)
                     /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"
     primer_bind     complement(4532..4551)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4597..4652
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4651..4670)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4740..4787
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4800..5505
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4888..5260
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4917..5505)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5514..5547)
                     /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(5715..6302)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5794..5813)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6315..7106,7107..7175))
                     /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     6938..6957
                     /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 ggggggsgrg 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 ccatgggcca
     2341 ccatcatcat catcatcacc accatcactc tgattacaag gatgacgacg ataagaccga
     2401 gaacctgtac ttccaatcca taagctagct tctcctcctg aaagatccat aacttcgtat
     2461 agcatacatt atacgaagtt atgcggccgc gacgtccaca tatacctgcc gttcactatt
     2521 atttagtgaa atgagatatt atgatatttt ctgaattgtg attaaaaagg caactttatg
     2581 cccatgcaac agaaactata aaaaatacag agaatgaaaa gaaacagata gattttttag
     2641 ttctttaggc ccgtagtctg caaatccttt tatgattttc tatcaaacaa aagaggaaaa
     2701 tagaccagtt gcaatccaaa cgagagtcta atagaatgag gtcgaaaagt aaatcgcgcg
     2761 ggtttgttac tgataaagca ggcaagacct aaaatgtgta aagggcaaag tgtatacttt
     2821 ggcgtcaccc cttacatatt ttaggtcttt ttttattgtg cgtaactaac ttgccatctt
     2881 caaacaggag ggctggaaga agcagaccgc taacacagta cataaaaaag gagacatgaa
     2941 cgatgaacat caaaaagttt gcaaaacaag caacagtatt aacctttact accgcactgc
     3001 tggcaggagg cgcaactcaa gcgtttgcga aagaaacgaa ccaaaagcca tataaggaaa
     3061 catacggcat ttcccatatt acacgccatg atatgctgca aatccctgaa cagcaaaaaa
     3121 atgaaaaata taaagttcct gagttcgatt cgtccacaat taaaaatatc tcttctgcaa
     3181 aaggcctgga cgtttgggac agctggccat tacaaaacac tgacggcact gtcgcaaact
     3241 atcacggcta ccacatcgtc tttgcattag ccggagatcc taaaaatgcg gatgacacat
     3301 cgatttacat gttctatcaa aaagtcggcg aaacttctat tgacagctgg aaaaacgctg
     3361 gccgcgtctt taaagacagc gacaaattcg atgcaaatga ttctatccta aaagaccaaa
     3421 cacaagaatg gtcaggttca gccacattta catctgacgg aaaaatccgt ttattctaca
     3481 ctgatttctc cggtaaacat tacggcaaac aaacactgac aactgcacaa gttaacgtat
     3541 cagcatcaga cagctctttg aacatcaacg gtgtagagga ttataaatca atctttgacg
     3601 gtgacggaaa aacgtatcaa aatgtacagc agttcatcga tgaaggcaac tacagctcag
     3661 gcgacaacca tacgctgaga gatcctcact acgtagaaga taaaggccac aaatacttag
     3721 tatttgaagc aaacactgga actgaagatg gctaccaagg cgaagaatct ttatttaaca
     3781 aagcatacta tggcaaaagc acatcattct tccgtcaaga aagtcaaaaa cttctgcaaa
     3841 gcgataaaaa acgcacggct gagttagcaa acggcgctct cggtatgatt gagctaaacg
     3901 atgattacac actgaaaaaa gtgatgaaac cgctgattgc atctaacaca gtaacagatg
     3961 aaattgaacg cgcgaacgtc tttaaaatga acggcaaatg gtacctgttc actgactccc
     4021 gcggatcaaa aatgacgatt gacggcatta cgtctaacga tatttacatg cttggttatg
     4081 tttctaattc tttaactggc ccatacaagc cgctgaacaa aactggcctt gtgttaaaaa
     4141 tggatcttga tcctaacgat gtaaccttta cttactcaca cttcgctgta cctcaagcga
     4201 aaggaaacaa tgtcgtgatt acaagctata tgacaaacag aggattctac gcagacaaac
     4261 aatcaacgtt tgcgcctagc ttcctgctga acatcaaagg caagaaaaca tctgttgtca
     4321 aagacagcat ccttgaacaa ggacaattaa cagttaacaa ataaaaacgc aaaagaaaat
     4381 gccgatatcc tattggcatt gacgtcaggt ggcacttttc gaggagttta ctagtaagta
     4441 aaggtggata cggcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa
     4501 tgccctggct cacaaatacc actgagatcg atctttttcc ctctgccaaa aattatgggg
     4561 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
     4621 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc
     4681 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgtaac
     4741 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaagcatgcg
     4801 gaggaaattc tccttgaagt ttccctggtg ttcaaagtaa aggagtttgc accagacgca
     4861 cctctgttca ctggtccggc gtattaaaac acgatacatt gttattagta catttattaa
     4921 gcgctagatt ctgtgcgttg ttgatttaca gacaattgtt gtacgtattt taataattca
     4981 ttaaatttat aatctttagg gtggtatgtt agagcgaaaa tcaaatgatt ttcagcgtct
     5041 ttatatctga atttaaatat taaatcctca atagatttgt aaaataggtt tcgattagtt
     5101 tcaaacaagg gttgtttttc cgaaccgatg gctggactat ctaatggatt ttcgctcaac
     5161 gccacaaaac ttgccaaatc ttgtagcagc aatctagctt tgtcgatatt cgtttgtgtt
     5221 ttgttttgta ataaaggttc gacgtcgttc aaaatattat gcgcttttgt atttctttca
     5281 tcactgtcgt tagtgtacaa ttgactcgac gtaaacacgt taaatagagc ttggacatat
     5341 ttaacatcgg gcgtgttagc tttattaggc cgattatcgt cgtcgtccca accctcgtcg
     5401 ttagaagttg cttccgaaga cgattttgcc atagccacac gacgcctatt aattgtgtcg
     5461 gctaacacgt ccgcgatcaa atttgtagtt gagctttttg gaattgcgat cgcataactt
     5521 cgtatagcat acattatacg aagttataag ctcggaacgc tgcgctcggt cgttcggctg
     5581 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5641 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5701 gcgttgctgg cgtttttcca taggctccgc cccctgacga gcatcacaaa aatcgacgct
     5761 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5821 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5881 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5941 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6001 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6061 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6121 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6181 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6241 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6301 aagaagatcc tttgttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6361 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6421 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6481 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6541 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6601 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6661 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6721 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6781 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6841 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6901 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6961 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7021 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7081 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7141 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7201 agcatttatc agggttattg tctcatgtcc gcgcgtt
//
