LOCUS       sequence-232313-        7305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shuttle vector for baculovirus production, using FlashBac bacmid; 
            for recombinant protein with N-terminal MBP tag, cleavable with 3C 
            and C-terminal mKate2, and HIS6, cleavable with 3C.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7305)
  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 7305)
  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: pOCC204
FEATURES             Location/Qualifiers
     source          1..7305
                     /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             1714..2409
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             2410..2433
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2443..2460
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2602..2736
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2639..2658)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2693..2712
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           2809..3181
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(3533..3550)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3643..4231)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3723..3742)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4402..5193,5194..5262))
                     /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     5025..5044
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5263..5367)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5668..5690
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5682..5699
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5683..5699
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5710..5729
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5710..5728
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6339..6463
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6501..7106)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     promoter        7214..7305
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     7230..7247
                     /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 ccgggatccg ctggctccgc tgctggttct ggcatggtga gcgagctgat taaggagaac
     1741 atgcacatga agctgtacat ggagggcacc gtgaacaacc accacttcaa gtgcacatcc
     1801 gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggc ggtcgagggc
     1861 ggccctctcc ccttcgcctt cgacatcctg gctaccagct tcatgtacgg cagcaaaacc
     1921 ttcatcaacc acacccaggg catccccgac ttctttaagc agtccttccc cgagggcttc
     1981 acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac ccaggacacc
     2041 agcctccagg acggctgcct catctacaac gtcaagatca gaggggtgaa cttcccatcc
     2101 aacggccctg tgatgcagaa gaaaacactc ggctgggagg cctccaccga gaccctgtac
     2161 cccgctgacg gcggcctgga aggcagagcc gacatggccc tgaagctcgt gggcgggggc
     2221 cacctgatct gcaacttgaa gaccacatac agatccaaga aacccgctaa gaacctcaag
     2281 atgcccggcg tctactatgt ggacagaaga ctggaaagaa tcaaggaggc cgacaaagag
     2341 acctacgtcg agcagcacga ggtggctgtg gccagatact gcgacctccc tagcaaactg
     2401 gggcacagac tggaagttct gttccagggg ccgggcagca gccatcacca tcaccatcac
     2461 agtggataat agaagcttgt cgagaagtac tagaggatca taatcagcca taccacattt
     2521 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2581 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     2641 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2701 tccaaactca tcaatgtatc ttatcatgtc tggatctgat cactgcttga gcctaggaga
     2761 tccgaaccag ataagtgaaa gcctgcatcc gcagatctta attaataaaa cacgatacat
     2821 tgttattagt acatttatta agcgctagat tctgtgcgtt gttgatttac agacaattgt
     2881 tgtacgtatt ttaataattc attaaattta taatctttag ggtggtatgt tagagcgaaa
     2941 atcaaatgat tttcagcgtc tttatatctg aatttaaata ttaaatcctc aatagatttg
     3001 taaaataggt ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat ggctggacta
     3061 tctaatggat tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag caatctagct
     3121 ttgtcgatat tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt caaaatatta
     3181 tgcgcttttg tatttctttc atcactgtcg ttagtgtaca attgactcga cgtaaacacg
     3241 ttaaataacg cttggacata tttaacatcg ggcgtgttag ctttattagg ccgattatcg
     3301 tcgtcgtccc aaccctcgtc gttagaagtt gcttccgaag acgattttgc catagccaca
     3361 cgacgcctat taattgtgtc ggctaacacg tccgcgatca aatttgtagt tgagcttttt
     3421 gggcctcatg ggccttccgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3481 tgcattaaca tggtcatagc tgtttccttg cgtattgggc gctctccgct tcctcgctca
     3541 ctgactcgct gcgctcggtc gttcgggtaa agcctggggt gcctaatgag caaaaggcca
     3601 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3661 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3721 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3781 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3841 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3901 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3961 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4021 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4081 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4141 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4201 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4261 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4321 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4381 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4441 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4501 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagaaccac gctcaccggc
     4561 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4621 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4681 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     4741 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4801 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4861 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4921 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4981 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5041 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5101 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5161 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5221 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5281 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5341 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     5401 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     5461 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     5521 agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt gggaagggcg
     5581 tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     5641 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     5701 agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca aggccgtgac
     5761 gttaaaacta ttaagccatc caatcgaccg ttagtcgaat caggaccgct ggtgcgagaa
     5821 gccgcgaagt atggcgaatg catcgtataa cgtgtggagt ccgctcatta gagcgtcatg
     5881 tttagacaag aaagctacat atttaattga tcccgatgat tttattgata aattgaccct
     5941 aactccatac acggtattct acaatggcgg ggttttggtc aaaatttccg gactgcgatt
     6001 gtacatgctg ttaacggctc cgcccactat taatgaaatt aaaaattcca attttaaaaa
     6061 acgcagcaag agaaacattt gtatgaaaga atgcgtagaa ggaaagaaaa atgtcgtgga
     6121 catgctgaac aacaagatta atatgcctcc gtgtataaaa aaaatattga acgatttgaa
     6181 agaaaacaat gtaccgcgcg gcggtatgta caggaagagg tttatactaa actgttacat
     6241 tgcaaacgtg gtttcgtgtg ccaagtgtga aaaccgatgt ttaatcaagg ctctgacgca
     6301 tttctacaac cacgactcca agtgtgtggg tgaagtcatg catcttttaa tcaaatccca
     6361 agatgtgtat aaaccaccaa actgccaaaa aatgaaaact gtggacaagc tctgtccgtt
     6421 tgctggcaac tgcaagggtc tcaatcctat ttgtaattat tgaataataa aacaattata
     6481 aatgctaaat ttgtttttta ttaacgatac aaaccaaacg caacaagaac atttgtagta
     6541 ttatctataa ttgaaaacgc gtagttataa tcgctgaggt aatatttaaa atcattttca
     6601 aatgattcac agttaatttg cgacaatata attttatttt cacataaact agacgccttg
     6661 tcgtcttctt cttcgtattc cttctctttt tcatttttct cctcataaaa attaacatag
     6721 ttattatcgt atccatatat gtatctatcg tatagagtaa attttttgtt gtcataaata
     6781 tatatgtctt ttttaatggg gtgtatagta ccgctgcgca tagtttttct gtaatttaca
     6841 acagtgctat tttctggtag ttcttcggag tgtgttgctt taattattaa atttatataa
     6901 tcaatgaatt tgggatcgtc ggttttgtac aatatgttgc cggcatagta cgcagcttct
     6961 tctagttcaa ttacaccatt ttttagcagc accggattaa cataactttc caaaatgttg
     7021 tacgaaccgt taaacaaaaa cagttcacct cccttttcta tactattgtc tgcgagcagt
     7081 tgtttgttgt taaaaataac agccattgta atgagacgca caaactaata tcacaagctg
     7141 ccaccatttt gttcgcccag gactctagct atagttctag tggttggcta cgtatactcc
     7201 ggaatattaa tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta
     7261 ttttactgtt ttcgtaacag ttttgtaata aaaaaaccta taaat
//
