LOCUS       sequence-224687-        6672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shuttle vector for baculovirus production, using FlashBac bacmid; 
            for rec. protein with N-terminal TwinStrep-MBP tag, cleavable with 
            TEV and C-terminal HIS6, cleavable with TEV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6672)
  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 6672)
  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: pOCC304
FEATURES             Location/Qualifiers
     source          1..6672
                     /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      211..220
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(223..228,229..252,253..288,289..312)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             325..1425
                     /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     1399..1422
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1477..1497
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1606..1911
                     /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     1790..1809
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1949..1969
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1979..1996
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2138..2272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2175..2194)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2229..2248
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           2345..2717
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(3069..3086)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3179..3767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3259..3278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3938..4729,4730..4798))
                     /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     4561..4580
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4800..4904)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5205..5227
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5219..5236
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5220..5236
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5247..5266
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5247..5265
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5876..6000
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6038..6643)
                     /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 attcggatta
      181 ttcataccgt cccaccatcg ggcgcggatc gccaccatgg gcagcgcttg gagccatccg
      241 cagtttgaaa aaggtggtgg tagcggtggt ggttcaggtg gtagtgcatg gtcacaccct
      301 cagtttgaga aaagcagcgg cagaatgaaa atcgaagaag gtaaactggt aatctggatt
      361 aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga gaaagatacc
      421 ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc acaggttgcg
      481 gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg tggctacgct
      541 caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa gctgtatccg
      601 tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat cgctgttgaa
      661 gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac ctgggaagag
      721 atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg
      781 caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc gttcaagtat
      841 gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc gaaagcgggt
      901 ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac cgattactcc
      961 atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg cccgtgggca
     1021 tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc gaccttcaag
     1081 ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg
     1141 aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga aggtctggaa
     1201 gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga ggaagagttg
     1261 gtgaaagatc cgcgtattgc cgccactatg gaaaacgccc agaaaggtga aatcatgccg
     1321 aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat caacgccgcc
     1381 agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactaattc gagctcgaac
     1441 aacaacaaca ataacaataa caacaacagc agcggcgaga acctatactt ccagtctgcg
     1501 gccgcaagcg ctatggccac aacctgttac attgcacaag ataaaaatat atcatcacga
     1561 acagtaaaac tgtctgctta cataaacagt aatacaaggg gtgttatgca gtttaaggtt
     1621 tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt
     1681 gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cccgtctgct gtcagataaa
     1741 gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc
     1801 accgatatgg ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac
     1861 cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc
     1921 tcccttatac acagccagtg gcgcgccgga gaacctatac ttccagggag gcagcagcca
     1981 tcaccatcac catcacagtg gataatagaa gcttgtcgag aagtactaga ggatcataat
     2041 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     2101 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     2161 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2221 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tctgatcact
     2281 gcttgagcct aggagatccg aaccagataa gtgaaagcct gcatccgcag atcttaatta
     2341 ataaaacacg atacattgtt attagtacat ttattaagcg ctagattctg tgcgttgttg
     2401 atttacagac aattgttgta cgtattttaa taattcatta aatttataat ctttagggtg
     2461 gtatgttaga gcgaaaatca aatgattttc agcgtcttta tatctgaatt taaatattaa
     2521 atcctcaata gatttgtaaa ataggtttcg attagtttca aacaagggtt gtttttccga
     2581 accgatggct ggactatcta atggattttc gctcaacgcc acaaaacttg ccaaatcttg
     2641 tagcagcaat ctagctttgt cgatattcgt ttgtgttttg ttttgtaata aaggttcgac
     2701 gtcgttcaaa atattatgcg cttttgtatt tctttcatca ctgtcgttag tgtacaattg
     2761 actcgacgta aacacgttaa ataacgcttg gacatattta acatcgggcg tgttagcttt
     2821 attaggccga ttatcgtcgt cgtcccaacc ctcgtcgtta gaagttgctt ccgaagacga
     2881 ttttgccata gccacacgac gcctattaat tgtgtcggct aacacgtccg cgatcaaatt
     2941 tgtagttgag ctttttgggc ctcatgggcc ttccgctcac tgcccgcttt ccagtcggga
     3001 aacctgtcgt gccagctgca ttaacatggt catagctgtt tccttgcgta ttgggcgctc
     3061 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc gggtaaagcc tggggtgcct
     3121 aatgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3181 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3241 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3301 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3361 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3421 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3481 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3541 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3601 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3661 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3721 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3781 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3841 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3901 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3961 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4021 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4081 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4141 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4201 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca
     4261 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4321 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4381 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4441 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4501 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg
     4561 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4621 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4681 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4741 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatta
     4801 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4861 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4921 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     4981 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     5041 agaccgagat agggttgagt ggccgctaca gggcgctccc attcgccatt caggctgcgc
     5101 aactgttggg aagggcgttt cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     5161 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     5221 taaaacgacg gccagtgagc gcgacgtaat acgactcact atagggcgaa ttggcggaag
     5281 gccgtcaagg ccgtgacgtt aaaactatta agccatccaa tcgaccgtta gtcgaatcag
     5341 gaccgctggt gcgagaagcc gcgaagtatg gcgaatgcat cgtataacgt gtggagtccg
     5401 ctcattagag cgtcatgttt agacaagaaa gctacatatt taattgatcc cgatgatttt
     5461 attgataaat tgaccctaac tccatacacg gtattctaca atggcggggt tttggtcaaa
     5521 atttccggac tgcgattgta catgctgtta acggctccgc ccactattaa tgaaattaaa
     5581 aattccaatt ttaaaaaacg cagcaagaga aacatttgta tgaaagaatg cgtagaagga
     5641 aagaaaaatg tcgtggacat gctgaacaac aagattaata tgcctccgtg tataaaaaaa
     5701 atattgaacg atttgaaaga aaacaatgta ccgcgcggcg gtatgtacag gaagaggttt
     5761 atactaaact gttacattgc aaacgtggtt tcgtgtgcca agtgtgaaaa ccgatgttta
     5821 atcaaggctc tgacgcattt ctacaaccac gactccaagt gtgtgggtga agtcatgcat
     5881 cttttaatca aatcccaaga tgtgtataaa ccaccaaact gccaaaaaat gaaaactgtg
     5941 gacaagctct gtccgtttgc tggcaactgc aagggtctca atcctatttg taattattga
     6001 ataataaaac aattataaat gctaaatttg ttttttatta acgatacaaa ccaaacgcaa
     6061 caagaacatt tgtagtatta tctataattg aaaacgcgta gttataatcg ctgaggtaat
     6121 atttaaaatc attttcaaat gattcacagt taatttgcga caatataatt ttattttcac
     6181 ataaactaga cgccttgtcg tcttcttctt cgtattcctt ctctttttca tttttctcct
     6241 cataaaaatt aacatagtta ttatcgtatc catatatgta tctatcgtat agagtaaatt
     6301 ttttgttgtc ataaatatat atgtcttttt taatggggtg tatagtaccg ctgcgcatag
     6361 tttttctgta atttacaaca gtgctatttt ctggtagttc ttcggagtgt gttgctttaa
     6421 ttattaaatt tatataatca atgaatttgg gatcgtcggt tttgtacaat atgttgccgg
     6481 catagtacgc agcttcttct agttcaatta caccattttt tagcagcacc ggattaacat
     6541 aactttccaa aatgttgtac gaaccgttaa acaaaaacag ttcacctccc ttttctatac
     6601 tattgtctgc gagcagttgt ttgttgttaa aaataacagc cattgtaatg agacgcacaa
     6661 actaatatca ca
//
