LOCUS       sequence-232361-        7314 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 C-terminal monomeric GFP, cleavable with TEV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7314)
  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 7314)
  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: pOCC119
FEATURES             Location/Qualifiers
     source          1..7314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..92
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     17..34
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     regulatory      134..143
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             152..169
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             182..1282
                     /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     1256..1279
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1337..1360
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1469..1774
                     /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     1653..1672
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1830..1850
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1851..1853,1854..1856,1857..2570)
                     /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(1896..1917)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2157..2176)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2504..2525
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2703..2837
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2740..2759)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2794..2813
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           2910..3282
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(3634..3651)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3744..4332)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3824..3843)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4503..5294,5295..5363))
                     /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     5126..5145
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5364..5468)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5769..5791
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5783..5800
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5784..5800
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5811..5830
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5811..5829
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6440..6564
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6602..7207)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
ORIGIN
        1 atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc
       61 gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca
      121 tcgggcgcgg atcgccacca tgggcagcag ccatcaccat caccatcaca gcagcggcag
      181 aatgaaaatc gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg
      241 tctcgctgaa gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca
      301 tccggataaa ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat
      361 tatcttctgg gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat
      421 caccccggac aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta
      481 caacggcaag ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa
      541 agatctgctg ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact
      601 gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc
      661 gctgattgct gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa
      721 agacgtgggc gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat
      781 taaaaacaaa cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa
      841 aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa
      901 agtgaattat ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt
      961 tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt
     1021 cctcgaaaac tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct
     1081 gggtgccgta gcgctgaagt cttacgagga agagttggtg aaagatccgc gtattgccgc
     1141 cactatggaa aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt
     1201 ctggtatgcc gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga
     1261 agccctgaaa gacgcgcaga ctaattcgag ctcgaacaac aacaacaata acaataacaa
     1321 caacagcagc ggcagactgg aagttctgtt ccaggggccg gcggccgcaa gcgctatggc
     1381 cacaacctgt tacattgcac aagataaaaa tatatcatca cgaacagtaa aactgtctgc
     1441 ttacataaac agtaatacaa ggggtgttat gcagtttaag gtttacacct ataaaagaga
     1501 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg
     1561 gatggtgatc cccctggcca gtgcccgtct gctgtcagat aaagtctccc gtgaacttta
     1621 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt
     1681 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa
     1741 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggctccctta tacacagcca
     1801 gtggcgcgcc gggatccagc agcggcagag aaaacttgta tttccagggc atggtgagca
     1861 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1921 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     1981 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2041 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     2101 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2161 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2221 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2281 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     2341 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     2401 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2461 cccagtccaa actgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     2521 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa tagaagcttg
     2581 tcgagaagta ctagaggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     2641 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     2701 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     2761 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     2821 cttatcatgt ctggatctga tcactgcttg agcctaggag atccgaacca gataagtgaa
     2881 agcctgcatc cgcagatctt aattaataaa acacgataca ttgttattag tacatttatt
     2941 aagcgctaga ttctgtgcgt tgttgattta cagacaattg ttgtacgtat tttaataatt
     3001 cattaaattt ataatcttta gggtggtatg ttagagcgaa aatcaaatga ttttcagcgt
     3061 ctttatatct gaatttaaat attaaatcct caatagattt gtaaaatagg tttcgattag
     3121 tttcaaacaa gggttgtttt tccgaaccga tggctggact atctaatgga ttttcgctca
     3181 acgccacaaa acttgccaaa tcttgtagca gcaatctagc tttgtcgata ttcgtttgtg
     3241 ttttgttttg taataaaggt tcgacgtcgt tcaaaatatt atgcgctttt gtatttcttt
     3301 catcactgtc gttagtgtac aattgactcg acgtaaacac gttaaataac gcttggacat
     3361 atttaacatc gggcgtgtta gctttattag gccgattatc gtcgtcgtcc caaccctcgt
     3421 cgttagaagt tgcttccgaa gacgattttg ccatagccac acgacgccta ttaattgtgt
     3481 cggctaacac gtccgcgatc aaatttgtag ttgagctttt tgggcctcat gggccttccg
     3541 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaac atggtcatag
     3601 ctgtttcctt gcgtattggg cgctctccgc ttcctcgctc actgactcgc tgcgctcggt
     3661 cgttcgggta aagcctgggg tgcctaatga gcaaaaggcc agcaaaaggc caggaaccgt
     3721 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3781 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3841 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3901 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3961 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4021 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4081 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4141 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4201 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4261 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4321 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4381 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4441 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4501 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4561 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4621 cccagtgctg caatgatacc gcgagaacca cgctcaccgg ctccagattt atcagcaata
     4681 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4741 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     4801 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     4861 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     4921 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     4981 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5041 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5101 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5161 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5221 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5281 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5341 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5401 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5461 gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa
     5521 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     5581 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtggccgc tacagggcgc
     5641 tcccattcgc cattcaggct gcgcaactgt tgggaagggc gtttcggtgc gggcctcttc
     5701 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     5761 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgacg taatacgact
     5821 cactataggg cgaattggcg gaaggccgtc aaggccgtga cgttaaaact attaagccat
     5881 ccaatcgacc gttagtcgaa tcaggaccgc tggtgcgaga agccgcgaag tatggcgaat
     5941 gcatcgtata acgtgtggag tccgctcatt agagcgtcat gtttagacaa gaaagctaca
     6001 tatttaattg atcccgatga ttttattgat aaattgaccc taactccata cacggtattc
     6061 tacaatggcg gggttttggt caaaatttcc ggactgcgat tgtacatgct gttaacggct
     6121 ccgcccacta ttaatgaaat taaaaattcc aattttaaaa aacgcagcaa gagaaacatt
     6181 tgtatgaaag aatgcgtaga aggaaagaaa aatgtcgtgg acatgctgaa caacaagatt
     6241 aatatgcctc cgtgtataaa aaaaatattg aacgatttga aagaaaacaa tgtaccgcgc
     6301 ggcggtatgt acaggaagag gtttatacta aactgttaca ttgcaaacgt ggtttcgtgt
     6361 gccaagtgtg aaaaccgatg tttaatcaag gctctgacgc atttctacaa ccacgactcc
     6421 aagtgtgtgg gtgaagtcat gcatctttta atcaaatccc aagatgtgta taaaccacca
     6481 aactgccaaa aaatgaaaac tgtggacaag ctctgtccgt ttgctggcaa ctgcaagggt
     6541 ctcaatccta tttgtaatta ttgaataata aaacaattat aaatgctaaa tttgtttttt
     6601 attaacgata caaaccaaac gcaacaagaa catttgtagt attatctata attgaaaacg
     6661 cgtagttata atcgctgagg taatatttaa aatcattttc aaatgattca cagttaattt
     6721 gcgacaatat aattttattt tcacataaac tagacgcctt gtcgtcttct tcttcgtatt
     6781 ccttctcttt ttcatttttc tcctcataaa aattaacata gttattatcg tatccatata
     6841 tgtatctatc gtatagagta aattttttgt tgtcataaat atatatgtct tttttaatgg
     6901 ggtgtatagt accgctgcgc atagtttttc tgtaatttac aacagtgcta ttttctggta
     6961 gttcttcgga gtgtgttgct ttaattatta aatttatata atcaatgaat ttgggatcgt
     7021 cggttttgta caatatgttg ccggcatagt acgcagcttc ttctagttca attacaccat
     7081 tttttagcag caccggatta acataacttt ccaaaatgtt gtacgaaccg ttaaacaaaa
     7141 acagttcacc tcccttttct atactattgt ctgcgagcag ttgtttgttg ttaaaaataa
     7201 cagccattgt aatgagacgc acaaactaat atcacaagct gccaccattt tgttcgccca
     7261 ggactctagc tatagttcta gtggttggct acgtatactc cggaatatta atag
//
