LOCUS       sequence-399465-        4634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Generate the baculovirus to express C-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 4634)
  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 4634)
  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-N-TEV-Twin-Strep
FEATURES             Location/Qualifiers
     source          1..4634
                     /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"
     3'UTR           2053..2425
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(2777..2794)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2887..3475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2967..2986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3646..4437,4438..4506))
                     /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     4269..4288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4507..4611)
                     /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 aagtgaagat
     2041 cttaattaat aaaacacgat acattgttat tagtacattt attaagcgct agattctgtg
     2101 cgttgttgat ttacagacaa ttgttgtacg tattttaata attcattaaa tttataatct
     2161 ttagggtggt atgttagagc gaaaatcaaa tgattttcag cgtctttata tctgaattta
     2221 aatattaaat cctcaataga tttgtaaaat aggtttcgat tagtttcaaa caagggttgt
     2281 ttttccgaac cgatggctgg actatctaat ggattttcgc tcaacgccac aaaacttgcc
     2341 aaatcttgta gcagcaatct agctttgtcg atattcgttt gtgttttgtt ttgtaataaa
     2401 ggttcgacgt cgttcaaaat attatgcgct tttgtatttc tttcatcact gtcgttagtg
     2461 tacaattgac tcgacgtaaa cacgttaaat aacgcttgga catatttaac atcgggcgtg
     2521 ttagctttat taggccgatt atcgtcgtcg tcccaaccct cgtcgttaga agttgcttcc
     2581 gaagacgatt ttgccatagc cacacgacgc ctattaattg tgtcggctaa cacgtccgcg
     2641 atcaaatttg tagttgagct ttttgggcct catgggcctt ccgctcactg cccgctttcc
     2701 agtcgggaaa cctgtcgtgc cagctgcatt aacatggtca tagctgtttc cttgcgtatt
     2761 gggcgctctc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg gtaaagcctg
     2821 gggtgcctaa tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2881 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2941 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3001 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3061 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3121 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3181 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3241 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3301 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3361 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3421 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3481 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3541 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3601 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3661 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3721 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3781 accgcgagaa ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3841 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3901 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3961 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4021 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4081 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4141 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4201 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4261 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4321 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4381 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4441 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4501 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4561 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4621 ccgaaaagtg ccac
//
