LOCUS       sequence-224693-        7384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shuttle vector for baculovirus production, using FlashBac bacmid; 
            for recombinant protein with N-terminal MBP, cleavable with 3C and 
            monomeric GFP and C-terminal TwinStrep, cleavable with 3C.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7384)
  AUTHORS   Lemaitre RP, Bogdanova A, Borgonovo B, Woodruff JB, Drechsel DN
  TITLE     FlexiBAC: a versatile, open-source baculovirus vector system for 
            protein expression, secretion, and proteolytic processing.
  JOURNAL   BMC Biotechnol. 2019 Mar 29;19(1):20. doi: 
            10.1186/s12896-019-0512-z.
  PUBMED    30925874
REFERENCE   2  (bases 1 to 7384)
  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: "BMC 
            Biotechnol."; date: "2019-03-29"; volume: "19(1):20. doi"; pages: " 
            10.1186/s12896-019-0512-z"
COMMENT     Sequence Label: pOCC193
FEATURES             Location/Qualifiers
     source          1..7384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        79..170
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     95..112
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     regulatory      212..221
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             224..1324
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1298..1321
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1379..1402
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1403..1405,1406..1408,1409..2119)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1448..1469)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1709..1728)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2056..2077
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2258..2563
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2442..2461
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2601..2624
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(2625..2630,2631..2654,2655..2690,2691..2714)
                     /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="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     polyA_signal    2850..2984
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2887..2906)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2941..2960
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           3057..3429
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(3781..3798)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3891..4479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3971..3990)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4650..5441,5442..5510))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5273..5292
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5512..5616)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5917..5939
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5931..5948
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5932..5948
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5959..5978
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5959..5977
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6588..6712
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6750..7355)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
ORIGIN
        1 agctgccacc attttgttcg cccaggactc tagctatagt tctagtggtt ggctacgtat
       61 actccggaat attaatagat catggagata attaaaatga taaccatctc gcaaataaat
      121 aagtatttta ctgttttcgt aacagttttg taataaaaaa acctataaat attccggatt
      181 attcataccg tcccaccatc gggcgcggat cgccaccatg ggcatgaaaa tcgaagaagg
      241 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
      301 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
      361 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
      421 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
      481 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
      541 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
      601 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
      661 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
      721 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
      781 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
      841 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
      901 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
      961 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     1021 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     1081 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     1141 gtcttacgag gaagagttgg tgaaagatcc gcgtattgcc gccactatgg aaaacgccca
     1201 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     1261 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     1321 gactaattcg agctcgaaca acaacaacaa taacaataac aacaacagca gcggcagact
     1381 ggaagttctg ttccaggggc cgatggtgag caagggcgag gagctgttca ccggggtggt
     1441 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     1501 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     1561 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     1621 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     1681 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     1741 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     1801 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     1861 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     1921 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     1981 cgtgctgctg cccgacaacc actacctgag cacccagtcc aaactgagca aagaccccaa
     2041 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     2101 catggacgag ctgtacaagg gatccgctgg ctccgctgct ggttctggcg cggccgcaag
     2161 cgctatggcc acaacctgtt acattgcaca agataaaaat atatcatcac gaacagtaaa
     2221 actgtctgct tacataaaca gtaatacaag gggtgttatg cagtttaagg tttacaccta
     2281 taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc
     2341 cgggcgacgg atggtgatcc ccctggccag tgcccgtctg ctgtcagata aagtctcccg
     2401 tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat
     2461 ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa
     2521 tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag gctcccttat
     2581 acacagccag tggcgcgccg ctggaagttc tgttccaggg gccgagcgct tggagccatc
     2641 cgcagtttga aaaaggtggt ggtagcggtg gtggttcagg tggtagtgca tggtcacacc
     2701 ctcagtttga gaaataatag aagcttgtcg agaagtacta gaggatcata atcagccata
     2761 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     2821 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     2881 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2941 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatctgatca ctgcttgagc
     3001 ctaggagatc cgaaccagat aagtgaaagc ctgcatccgc agatcttaat taataaaaca
     3061 cgatacattg ttattagtac atttattaag cgctagattc tgtgcgttgt tgatttacag
     3121 acaattgttg tacgtatttt aataattcat taaatttata atctttaggg tggtatgtta
     3181 gagcgaaaat caaatgattt tcagcgtctt tatatctgaa tttaaatatt aaatcctcaa
     3241 tagatttgta aaataggttt cgattagttt caaacaaggg ttgtttttcc gaaccgatgg
     3301 ctggactatc taatggattt tcgctcaacg ccacaaaact tgccaaatct tgtagcagca
     3361 atctagcttt gtcgatattc gtttgtgttt tgttttgtaa taaaggttcg acgtcgttca
     3421 aaatattatg cgcttttgta tttctttcat cactgtcgtt agtgtacaat tgactcgacg
     3481 taaacacgtt aaataacgct tggacatatt taacatcggg cgtgttagct ttattaggcc
     3541 gattatcgtc gtcgtcccaa ccctcgtcgt tagaagttgc ttccgaagac gattttgcca
     3601 tagccacacg acgcctatta attgtgtcgg ctaacacgtc cgcgatcaaa tttgtagttg
     3661 agctttttgg gcctcatggg ccttccgctc actgcccgct ttccagtcgg gaaacctgtc
     3721 gtgccagctg cattaacatg gtcatagctg tttccttgcg tattgggcgc tctccgcttc
     3781 ctcgctcact gactcgctgc gctcggtcgt tcgggtaaag cctggggtgc ctaatgagca
     3841 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3901 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3961 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4021 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4081 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4141 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4201 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4261 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4321 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4381 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4441 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4501 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4561 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4621 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4681 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4741 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4801 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4861 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4921 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg
     4981 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5041 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5101 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5161 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5221 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc
     5281 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5341 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5401 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5461 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat tactcttcct
     5521 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5581 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5641 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     5701 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     5761 atagggttga gtggccgcta cagggcgctc ccattcgcca ttcaggctgc gcaactgttg
     5821 ggaagggcgt ttcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     5881 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     5941 cggccagtga gcgcgacgta atacgactca ctatagggcg aattggcgga aggccgtcaa
     6001 ggccgtgacg ttaaaactat taagccatcc aatcgaccgt tagtcgaatc aggaccgctg
     6061 gtgcgagaag ccgcgaagta tggcgaatgc atcgtataac gtgtggagtc cgctcattag
     6121 agcgtcatgt ttagacaaga aagctacata tttaattgat cccgatgatt ttattgataa
     6181 attgacccta actccataca cggtattcta caatggcggg gttttggtca aaatttccgg
     6241 actgcgattg tacatgctgt taacggctcc gcccactatt aatgaaatta aaaattccaa
     6301 ttttaaaaaa cgcagcaaga gaaacatttg tatgaaagaa tgcgtagaag gaaagaaaaa
     6361 tgtcgtggac atgctgaaca acaagattaa tatgcctccg tgtataaaaa aaatattgaa
     6421 cgatttgaaa gaaaacaatg taccgcgcgg cggtatgtac aggaagaggt ttatactaaa
     6481 ctgttacatt gcaaacgtgg tttcgtgtgc caagtgtgaa aaccgatgtt taatcaaggc
     6541 tctgacgcat ttctacaacc acgactccaa gtgtgtgggt gaagtcatgc atcttttaat
     6601 caaatcccaa gatgtgtata aaccaccaaa ctgccaaaaa atgaaaactg tggacaagct
     6661 ctgtccgttt gctggcaact gcaagggtct caatcctatt tgtaattatt gaataataaa
     6721 acaattataa atgctaaatt tgttttttat taacgataca aaccaaacgc aacaagaaca
     6781 tttgtagtat tatctataat tgaaaacgcg tagttataat cgctgaggta atatttaaaa
     6841 tcattttcaa atgattcaca gttaatttgc gacaatataa ttttattttc acataaacta
     6901 gacgccttgt cgtcttcttc ttcgtattcc ttctcttttt catttttctc ctcataaaaa
     6961 ttaacatagt tattatcgta tccatatatg tatctatcgt atagagtaaa ttttttgttg
     7021 tcataaatat atatgtcttt tttaatgggg tgtatagtac cgctgcgcat agtttttctg
     7081 taatttacaa cagtgctatt ttctggtagt tcttcggagt gtgttgcttt aattattaaa
     7141 tttatataat caatgaattt gggatcgtcg gttttgtaca atatgttgcc ggcatagtac
     7201 gcagcttctt ctagttcaat tacaccattt tttagcagca ccggattaac ataactttcc
     7261 aaaatgttgt acgaaccgtt aaacaaaaac agttcacctc ccttttctat actattgtct
     7321 gcgagcagtt gtttgttgtt aaaaataaca gccattgtaa tgagacgcac aaactaatat
     7381 caca
//
