LOCUS       sequence-232366-        7317 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shuttle vector for baculovirus production, using FlashBac bacmid; 
            for rec. protein with N-terminal MBP tag, cleavable with 3C and 
            C-terminal mCherry, cleavable with TEV and HIS6, cleavable with 3C.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7317)
  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 7317)
  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: pOCC240
FEATURES             Location/Qualifiers
     source          1..7317
                     /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             54..1154
                     /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     1128..1151
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1209..1232
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1341..1646
                     /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     1525..1544
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1693..1713
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1714..2421
                     /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(1859..1877)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2127..2146
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2422..2445
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2455..2472
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2614..2748
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2651..2670)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2705..2724
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           2821..3193
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(3545..3562)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3655..4243)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3735..3754)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4414..5205,5206..5274))
                     /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     5037..5056
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5275..5379)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5680..5702
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5694..5711
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5695..5711
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5722..5741
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5722..5740
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6351..6475
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6513..7118)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     promoter        7226..7317
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     7242..7259
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
ORIGIN
        1 attccggatt attcataccg tcccaccatc gggcgcggat cgccaccatg ggcatgaaaa
       61 tcgaagaagg taaactggta atctggatta acggcgataa aggctataac ggtctcgctg
      121 aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata
      181 aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac attatcttct
      241 gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg
      301 acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca
      361 agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc
      421 tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga
      481 aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg
      541 ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg
      601 gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca
      661 aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa
      721 cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt
      781 atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc
      841 tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa
      901 actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg
      961 tagcgctgaa gtcttacgag gaagagttgg tgaaagatcc gcgtattgcc gccactatgg
     1021 aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg
     1081 ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga
     1141 aagacgcgca gactaattcg agctcgaaca acaacaacaa taacaataac aacaacagca
     1201 gcggcagact ggaagttctg ttccaggggc cggcggccgc aagcgctatg gccacaacct
     1261 gttacattgc acaagataaa aatatatcat cacgaacagt aaaactgtct gcttacataa
     1321 acagtaatac aaggggtgtt atgcagttta aggtttacac ctataaaaga gagagccgtt
     1381 atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga
     1441 tccccctggc cagtgcccgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg
     1501 tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct
     1561 ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca
     1621 ttaacctgat gttctgggga atataaatgt caggctccct tatacacagc cagtggcgcg
     1681 ccgggatcca gcgaaaactt gtatttccag ggcatggtga gcaagggcga ggaggataac
     1741 atggccatca tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc
     1801 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     1861 aagctgaagg tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag
     1921 ttcatgtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag
     1981 ctgtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg
     2041 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg
     2101 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagacgat gggctgggag
     2161 gcctcctccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg
     2221 ctgaagctga aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag
     2281 aagcccgtgc agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac
     2341 aacgaggact acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc
     2401 ggcatggacg agctgtacaa gctggaagtt ctgttccagg ggccgggcag cagccatcac
     2461 catcaccatc acagtggata atagaagctt gtcgagaagt actagaggat cataatcagc
     2521 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     2581 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     2641 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     2701 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatctg atcactgctt
     2761 gagcctagga gatccgaacc agataagtga aagcctgcat ccgcagatct taattaataa
     2821 aacacgatac attgttatta gtacatttat taagcgctag attctgtgcg ttgttgattt
     2881 acagacaatt gttgtacgta ttttaataat tcattaaatt tataatcttt agggtggtat
     2941 gttagagcga aaatcaaatg attttcagcg tctttatatc tgaatttaaa tattaaatcc
     3001 tcaatagatt tgtaaaatag gtttcgatta gtttcaaaca agggttgttt ttccgaaccg
     3061 atggctggac tatctaatgg attttcgctc aacgccacaa aacttgccaa atcttgtagc
     3121 agcaatctag ctttgtcgat attcgtttgt gttttgtttt gtaataaagg ttcgacgtcg
     3181 ttcaaaatat tatgcgcttt tgtatttctt tcatcactgt cgttagtgta caattgactc
     3241 gacgtaaaca cgttaaataa cgcttggaca tatttaacat cgggcgtgtt agctttatta
     3301 ggccgattat cgtcgtcgtc ccaaccctcg tcgttagaag ttgcttccga agacgatttt
     3361 gccatagcca cacgacgcct attaattgtg tcggctaaca cgtccgcgat caaatttgta
     3421 gttgagcttt ttgggcctca tgggccttcc gctcactgcc cgctttccag tcgggaaacc
     3481 tgtcgtgcca gctgcattaa catggtcata gctgtttcct tgcgtattgg gcgctctccg
     3541 cttcctcgct cactgactcg ctgcgctcgg tcgttcgggt aaagcctggg gtgcctaatg
     3601 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     3661 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     3721 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     3781 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     3841 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3901 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3961 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     4021 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     4081 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     4141 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     4201 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     4261 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     4321 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     4381 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     4441 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     4501 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaacc
     4561 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     4621 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     4681 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     4741 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     4801 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     4861 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4921 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4981 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     5041 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     5101 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     5161 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     5221 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     5281 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     5341 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     5401 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5461 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5521 gagatagggt tgagtggccg ctacagggcg ctcccattcg ccattcaggc tgcgcaactg
     5581 ttgggaaggg cgtttcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
     5641 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
     5701 cgacggccag tgagcgcgac gtaatacgac tcactatagg gcgaattggc ggaaggccgt
     5761 caaggccgtg acgttaaaac tattaagcca tccaatcgac cgttagtcga atcaggaccg
     5821 ctggtgcgag aagccgcgaa gtatggcgaa tgcatcgtat aacgtgtgga gtccgctcat
     5881 tagagcgtca tgtttagaca agaaagctac atatttaatt gatcccgatg attttattga
     5941 taaattgacc ctaactccat acacggtatt ctacaatggc ggggttttgg tcaaaatttc
     6001 cggactgcga ttgtacatgc tgttaacggc tccgcccact attaatgaaa ttaaaaattc
     6061 caattttaaa aaacgcagca agagaaacat ttgtatgaaa gaatgcgtag aaggaaagaa
     6121 aaatgtcgtg gacatgctga acaacaagat taatatgcct ccgtgtataa aaaaaatatt
     6181 gaacgatttg aaagaaaaca atgtaccgcg cggcggtatg tacaggaaga ggtttatact
     6241 aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat gtttaatcaa
     6301 ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca tgcatctttt
     6361 aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa ctgtggacaa
     6421 gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt attgaataat
     6481 aaaacaatta taaatgctaa atttgttttt tattaacgat acaaaccaaa cgcaacaaga
     6541 acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag gtaatattta
     6601 aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt ttcacataaa
     6661 ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt ctcctcataa
     6721 aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt aaattttttg
     6781 ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg catagttttt
     6841 ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc tttaattatt
     6901 aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt gccggcatag
     6961 tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt aacataactt
     7021 tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc tatactattg
     7081 tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg cacaaactaa
     7141 tatcacaagc tgccaccatt ttgttcgccc aggactctag ctatagttct agtggttggc
     7201 tacgtatact ccggaatatt aatagatcat ggagataatt aaaatgataa ccatctcgca
     7261 aataaataag tattttactg ttttcgtaac agttttgtaa taaaaaaacc tataaat
//
