LOCUS       sequence-224679-        7294 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shuttle vector for baculovirus production, using FlashBac bacmid; 
            for recombinant protein with N-terminal HIS6-MBP tag, cleavable with
            3C, and N-terminal mCherry, cleavable with TEV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7294)
  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 7294)
  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: pOCC254
FEATURES             Location/Qualifiers
     source          1..7294
                     /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             230..247
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             260..1360
                     /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     1334..1357
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1415..1438
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1439..2146
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1584..1602)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1852..1871
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2153..2173
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2282..2587
                     /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     2466..2485
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     polyA_signal    2760..2894
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2797..2816)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2851..2870
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           2967..3339
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(3691..3708)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3801..4389)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3881..3900)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4560..5351,5352..5420))
                     /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     5183..5202
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5422..5526)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5827..5849
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5841..5858
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5842..5858
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5869..5888
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5869..5887
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6498..6622
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6660..7265)
                     /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 ggcagcagcc atcaccatca
      241 ccatcacagc agcggcagaa tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg
      301 cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat
      361 taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac
      421 tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc
      481 tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac
      541 ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt
      601 atcgctgatt tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc
      661 ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga
      721 accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa
      781 cggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac
      841 cttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc
      901 agaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc
      961 caacatcgac accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca
     1021 accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa
     1081 agagctggca aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt
     1141 taataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggtgaa
     1201 agatccgcgt attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat
     1261 cccgcagatg tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg
     1321 tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cgaacaacaa
     1381 caacaataac aataacaaca acagcagcgg cagactggaa gttctgttcc aggggccgat
     1441 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     1501 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     1561 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     1621 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     1681 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     1741 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     1801 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     1861 gcagaagaag acgatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     1921 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     1981 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     2041 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     2101 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagggat ccgaaaactt
     2161 gtatttccag ggcgcggccg caagcgctat ggccacaacc tgttacattg cacaagataa
     2221 aaatatatca tcacgaacag taaaactgtc tgcttacata aacagtaata caaggggtgt
     2281 tatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt ttgtggatgt
     2341 acagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg ccagtgcccg
     2401 tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg gggatgaaag
     2461 ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg gggaagaagt
     2521 ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga tgttctgggg
     2581 aatataaatg tcaggctccc ttatacacag ccagtggcgc gccgtaatag aagcttgtcg
     2641 agaagtacta gaggatcata atcagccata ccacatttgt agaggtttta cttgctttaa
     2701 aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta
     2761 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2821 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2881 atcatgtctg gatctgatca ctgcttgagc ctaggagatc cgaaccagat aagtgaaagc
     2941 ctgcatccgc agatcttaat taataaaaca cgatacattg ttattagtac atttattaag
     3001 cgctagattc tgtgcgttgt tgatttacag acaattgttg tacgtatttt aataattcat
     3061 taaatttata atctttaggg tggtatgtta gagcgaaaat caaatgattt tcagcgtctt
     3121 tatatctgaa tttaaatatt aaatcctcaa tagatttgta aaataggttt cgattagttt
     3181 caaacaaggg ttgtttttcc gaaccgatgg ctggactatc taatggattt tcgctcaacg
     3241 ccacaaaact tgccaaatct tgtagcagca atctagcttt gtcgatattc gtttgtgttt
     3301 tgttttgtaa taaaggttcg acgtcgttca aaatattatg cgcttttgta tttctttcat
     3361 cactgtcgtt agtgtacaat tgactcgacg taaacacgtt aaataacgct tggacatatt
     3421 taacatcggg cgtgttagct ttattaggcc gattatcgtc gtcgtcccaa ccctcgtcgt
     3481 tagaagttgc ttccgaagac gattttgcca tagccacacg acgcctatta attgtgtcgg
     3541 ctaacacgtc cgcgatcaaa tttgtagttg agctttttgg gcctcatggg ccttccgctc
     3601 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaacatg gtcatagctg
     3661 tttccttgcg tattgggcgc tctccgcttc ctcgctcact gactcgctgc gctcggtcgt
     3721 tcgggtaaag cctggggtgc ctaatgagca aaaggccagc aaaaggccag gaaccgtaaa
     3781 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     3841 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     3901 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3961 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4021 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4081 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4141 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4201 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4261 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4321 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4381 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4441 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4501 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4561 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4621 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4681 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4741 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4801 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4861 gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4921 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4981 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5041 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5101 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5161 tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5221 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5281 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5341 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5401 cggaaatgtt gaatactcat tactcttcct ttttcaatat tattgaagca tttatcaggg
     5461 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     5521 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     5581 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     5641 atcccttata aatcaaaaga atagaccgag atagggttga gtggccgcta cagggcgctc
     5701 ccattcgcca ttcaggctgc gcaactgttg ggaagggcgt ttcggtgcgg gcctcttcgc
     5761 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     5821 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgacgta atacgactca
     5881 ctatagggcg aattggcgga aggccgtcaa ggccgtgacg ttaaaactat taagccatcc
     5941 aatcgaccgt tagtcgaatc aggaccgctg gtgcgagaag ccgcgaagta tggcgaatgc
     6001 atcgtataac gtgtggagtc cgctcattag agcgtcatgt ttagacaaga aagctacata
     6061 tttaattgat cccgatgatt ttattgataa attgacccta actccataca cggtattcta
     6121 caatggcggg gttttggtca aaatttccgg actgcgattg tacatgctgt taacggctcc
     6181 gcccactatt aatgaaatta aaaattccaa ttttaaaaaa cgcagcaaga gaaacatttg
     6241 tatgaaagaa tgcgtagaag gaaagaaaaa tgtcgtggac atgctgaaca acaagattaa
     6301 tatgcctccg tgtataaaaa aaatattgaa cgatttgaaa gaaaacaatg taccgcgcgg
     6361 cggtatgtac aggaagaggt ttatactaaa ctgttacatt gcaaacgtgg tttcgtgtgc
     6421 caagtgtgaa aaccgatgtt taatcaaggc tctgacgcat ttctacaacc acgactccaa
     6481 gtgtgtgggt gaagtcatgc atcttttaat caaatcccaa gatgtgtata aaccaccaaa
     6541 ctgccaaaaa atgaaaactg tggacaagct ctgtccgttt gctggcaact gcaagggtct
     6601 caatcctatt tgtaattatt gaataataaa acaattataa atgctaaatt tgttttttat
     6661 taacgataca aaccaaacgc aacaagaaca tttgtagtat tatctataat tgaaaacgcg
     6721 tagttataat cgctgaggta atatttaaaa tcattttcaa atgattcaca gttaatttgc
     6781 gacaatataa ttttattttc acataaacta gacgccttgt cgtcttcttc ttcgtattcc
     6841 ttctcttttt catttttctc ctcataaaaa ttaacatagt tattatcgta tccatatatg
     6901 tatctatcgt atagagtaaa ttttttgttg tcataaatat atatgtcttt tttaatgggg
     6961 tgtatagtac cgctgcgcat agtttttctg taatttacaa cagtgctatt ttctggtagt
     7021 tcttcggagt gtgttgcttt aattattaaa tttatataat caatgaattt gggatcgtcg
     7081 gttttgtaca atatgttgcc ggcatagtac gcagcttctt ctagttcaat tacaccattt
     7141 tttagcagca ccggattaac ataactttcc aaaatgttgt acgaaccgtt aaacaaaaac
     7201 agttcacctc ccttttctat actattgtct gcgagcagtt gtttgttgtt aaaaataaca
     7261 gccattgtaa tgagacgcac aaactaatat caca
//
