LOCUS       sequence-232286-        7293 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 7293)
  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 7293)
  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..7293
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      42..51
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             60..77
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             90..1190
                     /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     1164..1187
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1245..1268
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1269..1976
                     /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(1414..1432)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1682..1701
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1983..2003
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2112..2417
                     /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     2296..2315
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     polyA_signal    2590..2724
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2627..2646)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2681..2700
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           2797..3169
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(3521..3538)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3631..4219)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3711..3730)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4390..5181,5182..5250))
                     /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     5013..5032
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5251..5355)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5656..5678
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5670..5687
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5671..5687
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5698..5717
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5698..5716
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6327..6451
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6489..7094)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     promoter        7202..7293
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     7218..7235
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
ORIGIN
        1 attccggatt attcataccg tcccaccatc gggcgcggat cgccaccatg ggcagcagcc
       61 atcaccatca ccatcacagc agcggcagaa tgaaaatcga agaaggtaaa ctggtaatct
      121 ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag
      181 ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg
      241 ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct
      301 acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt
      361 atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg
      421 ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca aaaacctggg
      481 aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca
      541 acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca
      601 agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag
      661 cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt
      721 actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt
      781 gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta ctgccgacct
      841 tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca
      901 gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact gatgaaggtc
      961 tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag
     1021 agttggtgaa agatccgcgt attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca
     1081 tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg
     1141 ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact aattcgagct
     1201 cgaacaacaa caacaataac aataacaaca acagcagcgg cagactggaa gttctgttcc
     1261 aggggccgat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     1321 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     1381 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     1441 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     1501 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     1561 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     1621 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     1681 gccccgtaat gcagaagaag acgatgggct gggaggcctc ctccgagcgg atgtaccccg
     1741 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     1801 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     1861 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     1921 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagggat
     1981 ccgaaaactt gtatttccag ggcgcggccg caagcgctat ggccacaacc tgttacattg
     2041 cacaagataa aaatatatca tcacgaacag taaaactgtc tgcttacata aacagtaata
     2101 caaggggtgt tatgcagttt aaggtttaca cctataaaag agagagccgt tatcgtctgt
     2161 ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg atccccctgg
     2221 ccagtgcccg tctgctgtca gataaagtct cccgtgaact ttacccggtg gtgcatatcg
     2281 gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc tccgttatcg
     2341 gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc attaacctga
     2401 tgttctgggg aatataaatg tcaggctccc ttatacacag ccagtggcgc gccgtaatag
     2461 aagcttgtcg agaagtacta gaggatcata atcagccata ccacatttgt agaggtttta
     2521 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     2581 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     2641 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     2701 aatgtatctt atcatgtctg gatctgatca ctgcttgagc ctaggagatc cgaaccagat
     2761 aagtgaaagc ctgcatccgc agatcttaat taataaaaca cgatacattg ttattagtac
     2821 atttattaag cgctagattc tgtgcgttgt tgatttacag acaattgttg tacgtatttt
     2881 aataattcat taaatttata atctttaggg tggtatgtta gagcgaaaat caaatgattt
     2941 tcagcgtctt tatatctgaa tttaaatatt aaatcctcaa tagatttgta aaataggttt
     3001 cgattagttt caaacaaggg ttgtttttcc gaaccgatgg ctggactatc taatggattt
     3061 tcgctcaacg ccacaaaact tgccaaatct tgtagcagca atctagcttt gtcgatattc
     3121 gtttgtgttt tgttttgtaa taaaggttcg acgtcgttca aaatattatg cgcttttgta
     3181 tttctttcat cactgtcgtt agtgtacaat tgactcgacg taaacacgtt aaataacgct
     3241 tggacatatt taacatcggg cgtgttagct ttattaggcc gattatcgtc gtcgtcccaa
     3301 ccctcgtcgt tagaagttgc ttccgaagac gattttgcca tagccacacg acgcctatta
     3361 attgtgtcgg ctaacacgtc cgcgatcaaa tttgtagttg agctttttgg gcctcatggg
     3421 ccttccgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaacatg
     3481 gtcatagctg tttccttgcg tattgggcgc tctccgcttc ctcgctcact gactcgctgc
     3541 gctcggtcgt tcgggtaaag cctggggtgc ctaatgagca aaaggccagc aaaaggccag
     3601 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3661 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3721 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3781 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3841 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3901 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3961 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4021 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4081 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4141 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4201 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4261 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4321 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4381 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4441 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4501 atctggcccc agtgctgcaa tgataccgcg agaaccacgc tcaccggctc cagatttatc
     4561 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4621 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4681 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4741 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4801 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4861 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4921 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4981 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5041 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5101 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5161 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5221 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5281 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5341 aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag cgttaatatt
     5401 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     5461 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tggccgctac
     5521 agggcgctcc cattcgccat tcaggctgcg caactgttgg gaagggcgtt tcggtgcggg
     5581 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     5641 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgacgtaa
     5701 tacgactcac tatagggcga attggcggaa ggccgtcaag gccgtgacgt taaaactatt
     5761 aagccatcca atcgaccgtt agtcgaatca ggaccgctgg tgcgagaagc cgcgaagtat
     5821 ggcgaatgca tcgtataacg tgtggagtcc gctcattaga gcgtcatgtt tagacaagaa
     5881 agctacatat ttaattgatc ccgatgattt tattgataaa ttgaccctaa ctccatacac
     5941 ggtattctac aatggcgggg ttttggtcaa aatttccgga ctgcgattgt acatgctgtt
     6001 aacggctccg cccactatta atgaaattaa aaattccaat tttaaaaaac gcagcaagag
     6061 aaacatttgt atgaaagaat gcgtagaagg aaagaaaaat gtcgtggaca tgctgaacaa
     6121 caagattaat atgcctccgt gtataaaaaa aatattgaac gatttgaaag aaaacaatgt
     6181 accgcgcggc ggtatgtaca ggaagaggtt tatactaaac tgttacattg caaacgtggt
     6241 ttcgtgtgcc aagtgtgaaa accgatgttt aatcaaggct ctgacgcatt tctacaacca
     6301 cgactccaag tgtgtgggtg aagtcatgca tcttttaatc aaatcccaag atgtgtataa
     6361 accaccaaac tgccaaaaaa tgaaaactgt ggacaagctc tgtccgtttg ctggcaactg
     6421 caagggtctc aatcctattt gtaattattg aataataaaa caattataaa tgctaaattt
     6481 gttttttatt aacgatacaa accaaacgca acaagaacat ttgtagtatt atctataatt
     6541 gaaaacgcgt agttataatc gctgaggtaa tatttaaaat cattttcaaa tgattcacag
     6601 ttaatttgcg acaatataat tttattttca cataaactag acgccttgtc gtcttcttct
     6661 tcgtattcct tctctttttc atttttctcc tcataaaaat taacatagtt attatcgtat
     6721 ccatatatgt atctatcgta tagagtaaat tttttgttgt cataaatata tatgtctttt
     6781 ttaatggggt gtatagtacc gctgcgcata gtttttctgt aatttacaac agtgctattt
     6841 tctggtagtt cttcggagtg tgttgcttta attattaaat ttatataatc aatgaatttg
     6901 ggatcgtcgg ttttgtacaa tatgttgccg gcatagtacg cagcttcttc tagttcaatt
     6961 acaccatttt ttagcagcac cggattaaca taactttcca aaatgttgta cgaaccgtta
     7021 aacaaaaaca gttcacctcc cttttctata ctattgtctg cgagcagttg tttgttgtta
     7081 aaaataacag ccattgtaat gagacgcaca aactaatatc acaagctgcc accattttgt
     7141 tcgcccagga ctctagctat agttctagtg gttggctacg tatactccgg aatattaata
     7201 gatcatggag ataattaaaa tgataaccat ctcgcaaata aataagtatt ttactgtttt
     7261 cgtaacagtt ttgtaataaa aaaacctata aat
//
