LOCUS       sequence-232309-        7311 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 mCitrine, cleavable with TEV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7311)
  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 7311)
  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: pOCC231
FEATURES             Location/Qualifiers
     source          1..7311
                     /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             1377..1682
                     /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     1561..1580
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1738..1758
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1759..1761,1762..1764,1765..2478)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY*"
     primer_bind     complement(1804..1825)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2065..2084)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2412..2433
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2608..2742
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2645..2664)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2699..2718
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           2815..3187
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(3539..3556)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3649..4237)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3729..3748)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4408..5199,5200..5268))
                     /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     5031..5050
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5269..5373)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5674..5696
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5688..5705
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5689..5705
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5716..5735
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5716..5734
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6345..6469
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6507..7112)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     promoter        7220..7311
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     7236..7253
                     /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 aggggccggc ggccgcaagc gctatggcca caacctgtta cattgcacaa gataaaaata
     1321 tatcatcacg aacagtaaaa ctgtctgctt acataaacag taatacaagg ggtgttatgc
     1381 agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga
     1441 gtgatattat tgacacgccc gggcgacgga tggtgatccc cctggccagt gcccgtctgc
     1501 tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc
     1561 gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg
     1621 atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat
     1681 aaatgtcagg ctcccttata cacagccagt ggcgcgccgg gatccagcag cggcagagaa
     1741 aacttgtatt tccagggcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     1801 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     1861 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     1921 cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgatgtg cttcgcccgc
     1981 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     2041 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     2101 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     2161 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     2221 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     2281 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2341 ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga ccccaacgag
     2401 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     2461 gacgagctgt actaatagaa gcttgtcgag aagtactaga ggatcataat cagccatacc
     2521 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     2581 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     2641 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2701 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tctgatcact gcttgagcct
     2761 aggagatccg aaccagataa gtgaaagcct gcatccgcag atcttaatta ataaaacacg
     2821 atacattgtt attagtacat ttattaagcg ctagattctg tgcgttgttg atttacagac
     2881 aattgttgta cgtattttaa taattcatta aatttataat ctttagggtg gtatgttaga
     2941 gcgaaaatca aatgattttc agcgtcttta tatctgaatt taaatattaa atcctcaata
     3001 gatttgtaaa ataggtttcg attagtttca aacaagggtt gtttttccga accgatggct
     3061 ggactatcta atggattttc gctcaacgcc acaaaacttg ccaaatcttg tagcagcaat
     3121 ctagctttgt cgatattcgt ttgtgttttg ttttgtaata aaggttcgac gtcgttcaaa
     3181 atattatgcg cttttgtatt tctttcatca ctgtcgttag tgtacaattg actcgacgta
     3241 aacacgttaa ataacgcttg gacatattta acatcgggcg tgttagcttt attaggccga
     3301 ttatcgtcgt cgtcccaacc ctcgtcgtta gaagttgctt ccgaagacga ttttgccata
     3361 gccacacgac gcctattaat tgtgtcggct aacacgtccg cgatcaaatt tgtagttgag
     3421 ctttttgggc ctcatgggcc ttccgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3481 gccagctgca ttaacatggt catagctgtt tccttgcgta ttgggcgctc tccgcttcct
     3541 cgctcactga ctcgctgcgc tcggtcgttc gggtaaagcc tggggtgcct aatgagcaaa
     3601 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3661 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3721 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3781 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3841 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3901 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3961 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4021 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4081 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4141 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4201 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4261 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4321 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4381 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4441 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4501 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag aaccacgctc
     4561 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4621 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4681 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4741 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4801 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4861 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4921 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4981 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5041 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5101 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5161 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5221 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5281 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5341 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     5401 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     5461 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     5521 gggttgagtg gccgctacag ggcgctccca ttcgccattc aggctgcgca actgttggga
     5581 agggcgtttc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     5641 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
     5701 ccagtgagcg cgacgtaata cgactcacta tagggcgaat tggcggaagg ccgtcaaggc
     5761 cgtgacgtta aaactattaa gccatccaat cgaccgttag tcgaatcagg accgctggtg
     5821 cgagaagccg cgaagtatgg cgaatgcatc gtataacgtg tggagtccgc tcattagagc
     5881 gtcatgttta gacaagaaag ctacatattt aattgatccc gatgatttta ttgataaatt
     5941 gaccctaact ccatacacgg tattctacaa tggcggggtt ttggtcaaaa tttccggact
     6001 gcgattgtac atgctgttaa cggctccgcc cactattaat gaaattaaaa attccaattt
     6061 taaaaaacgc agcaagagaa acatttgtat gaaagaatgc gtagaaggaa agaaaaatgt
     6121 cgtggacatg ctgaacaaca agattaatat gcctccgtgt ataaaaaaaa tattgaacga
     6181 tttgaaagaa aacaatgtac cgcgcggcgg tatgtacagg aagaggttta tactaaactg
     6241 ttacattgca aacgtggttt cgtgtgccaa gtgtgaaaac cgatgtttaa tcaaggctct
     6301 gacgcatttc tacaaccacg actccaagtg tgtgggtgaa gtcatgcatc ttttaatcaa
     6361 atcccaagat gtgtataaac caccaaactg ccaaaaaatg aaaactgtgg acaagctctg
     6421 tccgtttgct ggcaactgca agggtctcaa tcctatttgt aattattgaa taataaaaca
     6481 attataaatg ctaaatttgt tttttattaa cgatacaaac caaacgcaac aagaacattt
     6541 gtagtattat ctataattga aaacgcgtag ttataatcgc tgaggtaata tttaaaatca
     6601 ttttcaaatg attcacagtt aatttgcgac aatataattt tattttcaca taaactagac
     6661 gccttgtcgt cttcttcttc gtattccttc tctttttcat ttttctcctc ataaaaatta
     6721 acatagttat tatcgtatcc atatatgtat ctatcgtata gagtaaattt tttgttgtca
     6781 taaatatata tgtctttttt aatggggtgt atagtaccgc tgcgcatagt ttttctgtaa
     6841 tttacaacag tgctattttc tggtagttct tcggagtgtg ttgctttaat tattaaattt
     6901 atataatcaa tgaatttggg atcgtcggtt ttgtacaata tgttgccggc atagtacgca
     6961 gcttcttcta gttcaattac accatttttt agcagcaccg gattaacata actttccaaa
     7021 atgttgtacg aaccgttaaa caaaaacagt tcacctccct tttctatact attgtctgcg
     7081 agcagttgtt tgttgttaaa aataacagcc attgtaatga gacgcacaaa ctaatatcac
     7141 aagctgccac cattttgttc gcccaggact ctagctatag ttctagtggt tggctacgta
     7201 tactccggaa tattaataga tcatggagat aattaaaatg ataaccatct cgcaaataaa
     7261 taagtatttt actgttttcg taacagtttt gtaataaaaa aacctataaa t
//
