LOCUS       sequence-399463-        4652 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Generate the baculovirus to express N-terminal Tobacco Etch Virus 
            (TEV) protease cleavable Twin-Strep-tagged protein 
            (ENLYFQGS-WSHPQFEK-(GGGS)2-GGSA-WSHPQFEK) in insect cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4652)
  AUTHORS   Furuta M, Murakami C, Numagami Y, Suzuki R, Sakane F
  TITLE     Diacylglycerol kinase zeta interacts with sphingomyelin synthase 1 
            and sphingomyelin synthase-related protein via different regions.
  JOURNAL   FEBS Open Bio. 2023 May 11. doi: 10.1002/2211-5463.13628.
  PUBMED    37166445
REFERENCE   2  (bases 1 to 4652)
  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: "FEBS Open 
            Bio. 2023 May 11. doi: 10.1002/2211-5463.13628."
COMMENT     Sequence Label: pOET3-C-TEV-Twin-Strep
FEATURES             Location/Qualifiers
     source          1..4652
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     278..300
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     292..309
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     293..309
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     320..339
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        320..338
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_recomb     949..1776
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             949..1073
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(1111..1716)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     CDS             1920..1940
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1950..1973,1974..2009,2010..2033)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             2034..2054
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     3'UTR           2071..2443
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(2795..2812)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2905..3493)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2985..3004)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3664..4455,4456..4524))
                     /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     4287..4306
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4525..4629)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt
      181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca
      361 aggccgtgac gttaaaacta ttaagccatc caatcgaccg ttagtcgaat caggaccgct
      421 ggtgcgagaa gccgcgaagt atggcgaatg catcgtataa cgtgtggagt ccgctcatta
      481 gagcgtcatg tttagacaag aaagctacat atttaattga tcccgatgat tttattgata
      541 aattgaccct aactccatac acggtattct acaatggcgg ggttttggtc aaaatttccg
      601 gactgcgatt gtacatgctg ttaacggctc cgcccactat taatgaaatt aaaaattcca
      661 attttaaaaa acgcagcaag agaaacattt gtatgaaaga atgcgtagaa ggaaagaaaa
      721 atgtcgtgga catgctgaac aacaagatta atatgcctcc gtgtataaaa aaaatattga
      781 acgatttgaa agaaaacaat gtaccgcgcg gcggtatgta caggaagagg tttatactaa
      841 actgttacat tgcaaacgtg gtttcgtgtg ccaagtgtga aaaccgatgt ttaatcaagg
      901 ctctgacgca tttctacaac cacgactcca agtgtgtggg tgaagtcatg catcttttaa
      961 tcaaatccca agatgtgtat aaaccaccaa actgccaaaa aatgaaaact gtggacaagc
     1021 tctgtccgtt tgctggcaac tgcaagggtc tcaatcctat ttgtaattat tgaataataa
     1081 aacaattata aatgctaaat ttgtttttta ttaacgatac aaaccaaacg caacaagaac
     1141 atttgtagta ttatctataa ttgaaaacgc gtagttataa tcgctgaggt aatatttaaa
     1201 atcattttca aatgattcac agttaatttg cgacaatata attttatttt cacataaact
     1261 agacgccttg tcgtcttctt cttcgtattc cttctctttt tcatttttct cctcataaaa
     1321 attaacatag ttattatcgt atccatatat gtatctatcg tatagagtaa attttttgtt
     1381 gtcataaata tatatgtctt ttttaatggg gtgtatagta ccgctgcgca tagtttttct
     1441 gtaatttaca acagtgctat tttctggtag ttcttcggag tgtgttgctt taattattaa
     1501 atttatataa tcaatgaatt tgggatcgtc ggttttgtac aatatgttgc cggcatagta
     1561 cgcagcttct tctagttcaa ttacaccatt ttttagcagc accggattaa cataactttc
     1621 caaaatgttg tacgaaccgt taaacaaaaa cagttcacct cccttttcta tactattgtc
     1681 tgcgagcagt tgtttgttgt taaaaataac agccattgta atgagacgca caaactaata
     1741 tcacaaactg gaaatgtcta tcaatatata gttgctgata tcgggagttc agtcgtcgaa
     1801 tgcaaagcgt aaaaaatatt aataaggtaa aaattacagc tacataaatt acacaattta
     1861 aacggatcca agcttctaga gtcgacgggc ccccgggctg cagaattcga gctctcgagg
     1921 aaaacctcta ttttcaaggc gccaccatgt ggtctcatcc ccagttcgaa aaaggtggtg
     1981 gaagtggtgg gggctcagga ggatcagcct ggtcacaccc tcagttcgag aaggaaaacc
     2041 tctattttca aggcagatct taattaataa aacacgatac attgttatta gtacatttat
     2101 taagcgctag attctgtgcg ttgttgattt acagacaatt gttgtacgta ttttaataat
     2161 tcattaaatt tataatcttt agggtggtat gttagagcga aaatcaaatg attttcagcg
     2221 tctttatatc tgaatttaaa tattaaatcc tcaatagatt tgtaaaatag gtttcgatta
     2281 gtttcaaaca agggttgttt ttccgaaccg atggctggac tatctaatgg attttcgctc
     2341 aacgccacaa aacttgccaa atcttgtagc agcaatctag ctttgtcgat attcgtttgt
     2401 gttttgtttt gtaataaagg ttcgacgtcg ttcaaaatat tatgcgcttt tgtatttctt
     2461 tcatcactgt cgttagtgta caattgactc gacgtaaaca cgttaaataa cgcttggaca
     2521 tatttaacat cgggcgtgtt agctttatta ggccgattat cgtcgtcgtc ccaaccctcg
     2581 tcgttagaag ttgcttccga agacgatttt gccatagcca cacgacgcct attaattgtg
     2641 tcggctaaca cgtccgcgat caaatttgta gttgagcttt ttgggcctca tgggccttcc
     2701 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa catggtcata
     2761 gctgtttcct tgcgtattgg gcgctctccg cttcctcgct cactgactcg ctgcgctcgg
     2821 tcgttcgggt aaagcctggg gtgcctaatg agcaaaaggc cagcaaaagg ccaggaaccg
     2881 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     2941 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3001 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3061 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3121 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3181 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3241 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3301 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3361 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3421 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3481 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3541 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3601 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3661 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3721 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3781 ccccagtgct gcaatgatac cgcgagaacc acgctcaccg gctccagatt tatcagcaat
     3841 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3901 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3961 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4021 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4081 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4141 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4201 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4261 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4321 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4381 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4441 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4501 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     4561 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4621 ggttccgcgc acatttcccc gaaaagtgcc ac
//
