LOCUS       sequence-224702-        7356 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 7356)
  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 7356)
  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..7356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        79..170
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     95..112
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     regulatory      212..221
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     sig_peptide     224..283
                     /codon_start=1
                     /product="signal sequence from honeybee melittin"
                     /label=melittin signal sequence
                     /translation="KFLVNVALVFMVVYISYIYA"
     CDS             290..1390
                     /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     1364..1387
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1445..1468
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1577..1882
                     /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     1761..1780
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             join(1944..1946,1947..1949,1950..2660)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1944..1946,1947..1949,1950..2660)
                     /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(1989..2010)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2250..2269)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2597..2618
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2670..2687
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2822..2956
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2859..2878)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2913..2932
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           3029..3401
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(3753..3770)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3863..4451)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3943..3962)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4622..5413,5414..5482))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5245..5264
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5484..5588)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5889..5911
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5903..5920
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5904..5920
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5931..5950
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5931..5949
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6560..6684
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6722..7327)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
ORIGIN
        1 agctgccacc attttgttcg cccaggactc tagctatagt tctagtggtt ggctacgtat
       61 actccggaat attaatagat catggagata attaaaatga taaccatctc gcaaataaat
      121 aagtatttta ctgttttcgt aacagttttg taataaaaaa acctataaat attccggatt
      181 attcataccg tcccaccatc gggcgcggat cgccaccatg ggcaaattct tagtcaacgt
      241 tgcccttgtt tttatggtcg tatacatttc ttacatctat gcgggatcca tgaaaatcga
      301 agaaggtaaa ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt
      361 cggtaagaaa ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact
      421 ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc
      481 acacgaccgc tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa
      541 agcgttccag gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct
      601 gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc
      661 gaacccgcca aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg
      721 taagagcgcg ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc
      781 tgacgggggt tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt
      841 ggataacgct ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca
      901 catgaatgca gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc
      961 gatgaccatc aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg
     1021 tgtaacggta ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag
     1081 cgcaggtatt aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta
     1141 tctgctgact gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc
     1201 gctgaagtct tacgaggaag agttggtgaa agatccgcgt attgccgcca ctatggaaaa
     1261 cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt
     1321 gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga
     1381 cgcgcagact aattcgagct cgaacaacaa caacaataac aataacaaca acagcagcgg
     1441 cagactggaa gttctgttcc aggggccggc ggccgcaagc gctatggcca caacctgtta
     1501 cattgcacaa gataaaaata tatcatcacg aacagtaaaa ctgtctgctt acataaacag
     1561 taatacaagg ggtgttatgc agtttaaggt ttacacctat aaaagagaga gccgttatcg
     1621 tctgtttgtg gatgtacaga gtgatattat tgacacgccc gggcgacgga tggtgatccc
     1681 cctggccagt gcccgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca
     1741 tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt
     1801 tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa
     1861 cctgatgttc tggggaatat aaatgtcagg ctcccttata cacagccagt ggcgcgccgg
     1921 ctggctccgc tgctggttct ggcatggtga gcaagggcga ggagctgttc accggggtgg
     1981 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2041 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2101 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     2161 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2221 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2281 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2341 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     2401 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2461 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2521 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     2581 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     2641 gcatggacga gctgtacaag agcagcggcc atcaccatca ccatcactaa agaagcttgt
     2701 cgagaagtac tagaggatca taatcagcca taccacattt gtagaggttt tacttgcttt
     2761 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     2821 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2881 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2941 ttatcatgtc tggatctgat cactgcttga gcctaggaga tccgaaccag ataagtgaaa
     3001 gcctgcatcc gcagatctta attaataaaa cacgatacat tgttattagt acatttatta
     3061 agcgctagat tctgtgcgtt gttgatttac agacaattgt tgtacgtatt ttaataattc
     3121 attaaattta taatctttag ggtggtatgt tagagcgaaa atcaaatgat tttcagcgtc
     3181 tttatatctg aatttaaata ttaaatcctc aatagatttg taaaataggt ttcgattagt
     3241 ttcaaacaag ggttgttttt ccgaaccgat ggctggacta tctaatggat tttcgctcaa
     3301 cgccacaaaa cttgccaaat cttgtagcag caatctagct ttgtcgatat tcgtttgtgt
     3361 tttgttttgt aataaaggtt cgacgtcgtt caaaatatta tgcgcttttg tatttctttc
     3421 atcactgtcg ttagtgtaca attgactcga cgtaaacacg ttaaataacg cttggacata
     3481 tttaacatcg ggcgtgttag ctttattagg ccgattatcg tcgtcgtccc aaccctcgtc
     3541 gttagaagtt gcttccgaag acgattttgc catagccaca cgacgcctat taattgtgtc
     3601 ggctaacacg tccgcgatca aatttgtagt tgagcttttt gggcctcatg ggccttccgc
     3661 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaaca tggtcatagc
     3721 tgtttccttg cgtattgggc gctctccgct tcctcgctca ctgactcgct gcgctcggtc
     3781 gttcgggtaa agcctggggt gcctaatgag caaaaggcca gcaaaaggcc aggaaccgta
     3841 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3901 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3961 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4021 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4081 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4141 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4201 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4261 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4321 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4381 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4441 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4501 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     4561 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     4621 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     4681 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     4741 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     4801 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     4861 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4921 acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4981 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5041 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5101 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5161 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5221 gctcttgccc ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5281 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5341 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5401 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5461 cacggaaatg ttgaatactc attactcttc ctttttcaat attattgaag catttatcag
     5521 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5581 gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa
     5641 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     5701 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtggccgc tacagggcgc
     5761 tcccattcgc cattcaggct gcgcaactgt tgggaagggc gtttcggtgc gggcctcttc
     5821 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     5881 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgacg taatacgact
     5941 cactataggg cgaattggcg gaaggccgtc aaggccgtga cgttaaaact attaagccat
     6001 ccaatcgacc gttagtcgaa tcaggaccgc tggtgcgaga agccgcgaag tatggcgaat
     6061 gcatcgtata acgtgtggag tccgctcatt agagcgtcat gtttagacaa gaaagctaca
     6121 tatttaattg atcccgatga ttttattgat aaattgaccc taactccata cacggtattc
     6181 tacaatggcg gggttttggt caaaatttcc ggactgcgat tgtacatgct gttaacggct
     6241 ccgcccacta ttaatgaaat taaaaattcc aattttaaaa aacgcagcaa gagaaacatt
     6301 tgtatgaaag aatgcgtaga aggaaagaaa aatgtcgtgg acatgctgaa caacaagatt
     6361 aatatgcctc cgtgtataaa aaaaatattg aacgatttga aagaaaacaa tgtaccgcgc
     6421 ggcggtatgt acaggaagag gtttatacta aactgttaca ttgcaaacgt ggtttcgtgt
     6481 gccaagtgtg aaaaccgatg tttaatcaag gctctgacgc atttctacaa ccacgactcc
     6541 aagtgtgtgg gtgaagtcat gcatctttta atcaaatccc aagatgtgta taaaccacca
     6601 aactgccaaa aaatgaaaac tgtggacaag ctctgtccgt ttgctggcaa ctgcaagggt
     6661 ctcaatccta tttgtaatta ttgaataata aaacaattat aaatgctaaa tttgtttttt
     6721 attaacgata caaaccaaac gcaacaagaa catttgtagt attatctata attgaaaacg
     6781 cgtagttata atcgctgagg taatatttaa aatcattttc aaatgattca cagttaattt
     6841 gcgacaatat aattttattt tcacataaac tagacgcctt gtcgtcttct tcttcgtatt
     6901 ccttctcttt ttcatttttc tcctcataaa aattaacata gttattatcg tatccatata
     6961 tgtatctatc gtatagagta aattttttgt tgtcataaat atatatgtct tttttaatgg
     7021 ggtgtatagt accgctgcgc atagtttttc tgtaatttac aacagtgcta ttttctggta
     7081 gttcttcgga gtgtgttgct ttaattatta aatttatata atcaatgaat ttgggatcgt
     7141 cggttttgta caatatgttg ccggcatagt acgcagcttc ttctagttca attacaccat
     7201 tttttagcag caccggatta acataacttt ccaaaatgtt gtacgaaccg ttaaacaaaa
     7261 acagttcacc tcccttttct atactattgt ctgcgagcag ttgtttgttg ttaaaaataa
     7321 cagccattgt aatgagacgc acaaactaat atcaca
//
