LOCUS       sequence-232258-        7355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shuttle vector for baculovirus production, using FlashBac bacmid; 
            for recombinant protein with honey bee melitine secretion signal, 
            N-terminal MBP, cleavable with 3C protease, and C-terminal 
            eGFP-HIS6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7355)
  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 7355)
  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: pOCC94
FEATURES             Location/Qualifiers
     source          1..7355
                     /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"
     sig_peptide     54..113
                     /codon_start=1
                     /product="signal sequence from honeybee melittin"
                     /label=melittin signal sequence
                     /translation="KFLVNVALVFMVVYISYIYA"
     CDS             120..1220
                     /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     1194..1217
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1275..1298
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1407..1712
                     /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     1591..1610
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             join(1774..1776,1777..1779,1780..2490)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1774..1776,1777..1779,1780..2490)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1819..1840)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2080..2099)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2427..2448
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2500..2517
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2652..2786
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2689..2708)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2743..2762
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           2859..3231
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(3583..3600)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3693..4281)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3773..3792)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4452..5243,5244..5312))
                     /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     5075..5094
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5313..5417)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5718..5740
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5732..5749
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5733..5749
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5760..5779
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5760..5778
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6389..6513
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6551..7156)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     promoter        7264..7355
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     7280..7297
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
ORIGIN
        1 attccggatt attcataccg tcccaccatc gggcgcggat cgccaccatg ggcaaattct
       61 tagtcaacgt tgcccttgtt tttatggtcg tatacatttc ttacatctat gcgggatcca
      121 tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc
      181 tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc
      241 cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta
      301 tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca
      361 ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca
      421 acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag
      481 atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga
      541 aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc
      601 tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag
      661 acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta
      721 aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag
      781 gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag
      841 tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg
      901 gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc
      961 tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg
     1021 gtgccgtagc gctgaagtct tacgaggaag agttggtgaa agatccgcgt attgccgcca
     1081 ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct
     1141 ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag
     1201 ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacaataac aataacaaca
     1261 acagcagcgg cagactggaa gttctgttcc aggggccggc ggccgcaagc gctatggcca
     1321 caacctgtta cattgcacaa gataaaaata tatcatcacg aacagtaaaa ctgtctgctt
     1381 acataaacag taatacaagg ggtgttatgc agtttaaggt ttacacctat aaaagagaga
     1441 gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccc gggcgacgga
     1501 tggtgatccc cctggccagt gcccgtctgc tgtcagataa agtctcccgt gaactttacc
     1561 cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc
     1621 cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa
     1681 acgccattaa cctgatgttc tggggaatat aaatgtcagg ctcccttata cacagccagt
     1741 ggcgcgccgg ctggctccgc tgctggttct ggcatggtga gcaagggcga ggagctgttc
     1801 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1861 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1921 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     1981 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     2041 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     2101 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2161 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     2221 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     2281 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     2341 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     2401 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2461 atcactctcg gcatggacga gctgtacaag agcagcggcc atcaccatca ccatcactaa
     2521 agaagcttgt cgagaagtac tagaggatca taatcagcca taccacattt gtagaggttt
     2581 tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa
     2641 ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2701 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2761 tcaatgtatc ttatcatgtc tggatctgat cactgcttga gcctaggaga tccgaaccag
     2821 ataagtgaaa gcctgcatcc gcagatctta attaataaaa cacgatacat tgttattagt
     2881 acatttatta agcgctagat tctgtgcgtt gttgatttac agacaattgt tgtacgtatt
     2941 ttaataattc attaaattta taatctttag ggtggtatgt tagagcgaaa atcaaatgat
     3001 tttcagcgtc tttatatctg aatttaaata ttaaatcctc aatagatttg taaaataggt
     3061 ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat ggctggacta tctaatggat
     3121 tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag caatctagct ttgtcgatat
     3181 tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt caaaatatta tgcgcttttg
     3241 tatttctttc atcactgtcg ttagtgtaca attgactcga cgtaaacacg ttaaataacg
     3301 cttggacata tttaacatcg ggcgtgttag ctttattagg ccgattatcg tcgtcgtccc
     3361 aaccctcgtc gttagaagtt gcttccgaag acgattttgc catagccaca cgacgcctat
     3421 taattgtgtc ggctaacacg tccgcgatca aatttgtagt tgagcttttt gggcctcatg
     3481 ggccttccgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaaca
     3541 tggtcatagc tgtttccttg cgtattgggc gctctccgct tcctcgctca ctgactcgct
     3601 gcgctcggtc gttcgggtaa agcctggggt gcctaatgag caaaaggcca gcaaaaggcc
     3661 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3721 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3781 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3841 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     3901 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     3961 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4021 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4081 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4141 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4201 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4261 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4321 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4381 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4441 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4501 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4561 ccatctggcc ccagtgctgc aatgataccg cgagaaccac gctcaccggc tccagattta
     4621 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     4681 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     4741 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     4801 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     4861 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     4921 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     4981 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5041 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5101 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5161 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5221 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5281 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5341 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5401 caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata
     5461 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     5521 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtggccgct
     5581 acagggcgct cccattcgcc attcaggctg cgcaactgtt gggaagggcg tttcggtgcg
     5641 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     5701 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgacgt
     5761 aatacgactc actatagggc gaattggcgg aaggccgtca aggccgtgac gttaaaacta
     5821 ttaagccatc caatcgaccg ttagtcgaat caggaccgct ggtgcgagaa gccgcgaagt
     5881 atggcgaatg catcgtataa cgtgtggagt ccgctcatta gagcgtcatg tttagacaag
     5941 aaagctacat atttaattga tcccgatgat tttattgata aattgaccct aactccatac
     6001 acggtattct acaatggcgg ggttttggtc aaaatttccg gactgcgatt gtacatgctg
     6061 ttaacggctc cgcccactat taatgaaatt aaaaattcca attttaaaaa acgcagcaag
     6121 agaaacattt gtatgaaaga atgcgtagaa ggaaagaaaa atgtcgtgga catgctgaac
     6181 aacaagatta atatgcctcc gtgtataaaa aaaatattga acgatttgaa agaaaacaat
     6241 gtaccgcgcg gcggtatgta caggaagagg tttatactaa actgttacat tgcaaacgtg
     6301 gtttcgtgtg ccaagtgtga aaaccgatgt ttaatcaagg ctctgacgca tttctacaac
     6361 cacgactcca agtgtgtggg tgaagtcatg catcttttaa tcaaatccca agatgtgtat
     6421 aaaccaccaa actgccaaaa aatgaaaact gtggacaagc tctgtccgtt tgctggcaac
     6481 tgcaagggtc tcaatcctat ttgtaattat tgaataataa aacaattata aatgctaaat
     6541 ttgtttttta ttaacgatac aaaccaaacg caacaagaac atttgtagta ttatctataa
     6601 ttgaaaacgc gtagttataa tcgctgaggt aatatttaaa atcattttca aatgattcac
     6661 agttaatttg cgacaatata attttatttt cacataaact agacgccttg tcgtcttctt
     6721 cttcgtattc cttctctttt tcatttttct cctcataaaa attaacatag ttattatcgt
     6781 atccatatat gtatctatcg tatagagtaa attttttgtt gtcataaata tatatgtctt
     6841 ttttaatggg gtgtatagta ccgctgcgca tagtttttct gtaatttaca acagtgctat
     6901 tttctggtag ttcttcggag tgtgttgctt taattattaa atttatataa tcaatgaatt
     6961 tgggatcgtc ggttttgtac aatatgttgc cggcatagta cgcagcttct tctagttcaa
     7021 ttacaccatt ttttagcagc accggattaa cataactttc caaaatgttg tacgaaccgt
     7081 taaacaaaaa cagttcacct cccttttcta tactattgtc tgcgagcagt tgtttgttgt
     7141 taaaaataac agccattgta atgagacgca caaactaata tcacaagctg ccaccatttt
     7201 gttcgcccag gactctagct atagttctag tggttggcta cgtatactcc ggaatattaa
     7261 tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt
     7321 ttcgtaacag ttttgtaata aaaaaaccta taaat
//
