LOCUS       sequence-224686-        7165 bp    DNA     circular SYN 15-APR-2025
DEFINITION  shuttle vector for baculovirus production, using FlashBac bacmid; 
            for rec. protein with N-terminal MBP tag, cleavable with 3C and 
            C-terminal SNAP, cleavable with TEV and HIS6, cleavable with 3C.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7165)
  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 7165)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pOCC210
FEATURES             Location/Qualifiers
     source          1..7165
                     /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"
     CDS             224..1324
                     /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     1298..1321
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1379..1402
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1511..1816
                     /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     1695..1714
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             1872..1892
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1893..2438
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             2439..2462
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2472..2489
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2631..2765
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2668..2687)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2722..2741
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           2838..3210
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(3562..3579)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3672..4260)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3752..3771)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4431..5222,5223..5291))
                     /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     5054..5073
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5293..5397)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5698..5720
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5712..5729
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5713..5729
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5740..5759
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5740..5758
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6369..6493
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6531..7136)
                     /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 ggcatgaaaa tcgaagaagg
      241 taaactggta atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa
      301 gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata aactggaaga
      361 gaaattccca caggttgcgg caactggcga tggccctgac attatcttct gggcacacga
      421 ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt
      481 ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc
      541 ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc
      601 gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag
      661 cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg
      721 gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa
      781 cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa
      841 tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac
      901 catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac
      961 ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg
     1021 tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct
     1081 gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa
     1141 gtcttacgag gaagagttgg tgaaagatcc gcgtattgcc gccactatgg aaaacgccca
     1201 gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac
     1261 tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca
     1321 gactaattcg agctcgaaca acaacaacaa taacaataac aacaacagca gcggcagact
     1381 ggaagttctg ttccaggggc cggcggccgc aagcgctatg gccacaacct gttacattgc
     1441 acaagataaa aatatatcat cacgaacagt aaaactgtct gcttacataa acagtaatac
     1501 aaggggtgtt atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt
     1561 tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc
     1621 cagtgcccgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg
     1681 ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg
     1741 ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat
     1801 gttctgggga atataaatgt caggctccct tatacacagc cagtggcgcg ccgggatcca
     1861 gcagcggcag agaaaacttg tatttccagg gcatggacaa agactgcgaa atgaagcgca
     1921 ccaccctgga tagccctctg ggcaagctgg aactgtctgg gtgcgaacag ggcctgcacc
     1981 gtatcatctt cctgggcaaa ggaacatctg ccgccgacgc cgtggaagtg cctgccccag
     2041 ccgccgtgct gggcggacca gagccactga tgcaggccac cgcctggctc aacgcctact
     2101 ttcaccagcc tgaggccatc gaggagttcc ctgtgccagc cctgcaccac ccagtgttcc
     2161 agcaggagag ctttacccgc caggtgctgt ggaaactgct gaaagtggtg aagttcggag
     2221 aggtcatcag ctacagccac ctggccgccc tggccggcaa tcccgccgcc accgccgccg
     2281 tgaaaaccgc cctgagcgga aatcccgtgc ccattctgat cccctgccac cgggtggtgc
     2341 agggcgacct ggacgtgggg ggctacgagg gcgggctcgc cgtgaaagag tggctgctgg
     2401 cccacgaggg ccacagactg ggcaagcctg ggctgggtct ggaagttctg ttccaggggc
     2461 cgggcagcag ccatcaccat caccatcaca gtggataata gaagcttgtc gagaagtact
     2521 agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     2581 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     2641 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     2701 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     2761 ggatctgatc actgcttgag cctaggagat ccgaaccaga taagtgaaag cctgcatccg
     2821 cagatcttaa ttaataaaac acgatacatt gttattagta catttattaa gcgctagatt
     2881 ctgtgcgttg ttgatttaca gacaattgtt gtacgtattt taataattca ttaaatttat
     2941 aatctttagg gtggtatgtt agagcgaaaa tcaaatgatt ttcagcgtct ttatatctga
     3001 atttaaatat taaatcctca atagatttgt aaaataggtt tcgattagtt tcaaacaagg
     3061 gttgtttttc cgaaccgatg gctggactat ctaatggatt ttcgctcaac gccacaaaac
     3121 ttgccaaatc ttgtagcagc aatctagctt tgtcgatatt cgtttgtgtt ttgttttgta
     3181 ataaaggttc gacgtcgttc aaaatattat gcgcttttgt atttctttca tcactgtcgt
     3241 tagtgtacaa ttgactcgac gtaaacacgt taaataacgc ttggacatat ttaacatcgg
     3301 gcgtgttagc tttattaggc cgattatcgt cgtcgtccca accctcgtcg ttagaagttg
     3361 cttccgaaga cgattttgcc atagccacac gacgcctatt aattgtgtcg gctaacacgt
     3421 ccgcgatcaa atttgtagtt gagctttttg ggcctcatgg gccttccgct cactgcccgc
     3481 tttccagtcg ggaaacctgt cgtgccagct gcattaacat ggtcatagct gtttccttgc
     3541 gtattgggcg ctctccgctt cctcgctcac tgactcgctg cgctcggtcg ttcgggtaaa
     3601 gcctggggtg cctaatgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3661 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3721 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3781 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3841 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3901 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3961 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4021 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4081 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4141 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4201 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4261 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4321 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4381 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4441 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4501 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4561 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4621 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4681 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4741 attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4801 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4861 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4921 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4981 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5041 gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5101 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5161 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5221 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5281 tgaatactca ttactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5341 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5401 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta
     5461 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     5521 aaatcaaaag aatagaccga gatagggttg agtggccgct acagggcgct cccattcgcc
     5581 attcaggctg cgcaactgtt gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc
     5641 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     5701 agtcacgacg ttgtaaaacg acggccagtg agcgcgacgt aatacgactc actatagggc
     5761 gaattggcgg aaggccgtca aggccgtgac gttaaaacta ttaagccatc caatcgaccg
     5821 ttagtcgaat caggaccgct ggtgcgagaa gccgcgaagt atggcgaatg catcgtataa
     5881 cgtgtggagt ccgctcatta gagcgtcatg tttagacaag aaagctacat atttaattga
     5941 tcccgatgat tttattgata aattgaccct aactccatac acggtattct acaatggcgg
     6001 ggttttggtc aaaatttccg gactgcgatt gtacatgctg ttaacggctc cgcccactat
     6061 taatgaaatt aaaaattcca attttaaaaa acgcagcaag agaaacattt gtatgaaaga
     6121 atgcgtagaa ggaaagaaaa atgtcgtgga catgctgaac aacaagatta atatgcctcc
     6181 gtgtataaaa aaaatattga acgatttgaa agaaaacaat gtaccgcgcg gcggtatgta
     6241 caggaagagg tttatactaa actgttacat tgcaaacgtg gtttcgtgtg ccaagtgtga
     6301 aaaccgatgt ttaatcaagg ctctgacgca tttctacaac cacgactcca agtgtgtggg
     6361 tgaagtcatg catcttttaa tcaaatccca agatgtgtat aaaccaccaa actgccaaaa
     6421 aatgaaaact gtggacaagc tctgtccgtt tgctggcaac tgcaagggtc tcaatcctat
     6481 ttgtaattat tgaataataa aacaattata aatgctaaat ttgtttttta ttaacgatac
     6541 aaaccaaacg caacaagaac atttgtagta ttatctataa ttgaaaacgc gtagttataa
     6601 tcgctgaggt aatatttaaa atcattttca aatgattcac agttaatttg cgacaatata
     6661 attttatttt cacataaact agacgccttg tcgtcttctt cttcgtattc cttctctttt
     6721 tcatttttct cctcataaaa attaacatag ttattatcgt atccatatat gtatctatcg
     6781 tatagagtaa attttttgtt gtcataaata tatatgtctt ttttaatggg gtgtatagta
     6841 ccgctgcgca tagtttttct gtaatttaca acagtgctat tttctggtag ttcttcggag
     6901 tgtgttgctt taattattaa atttatataa tcaatgaatt tgggatcgtc ggttttgtac
     6961 aatatgttgc cggcatagta cgcagcttct tctagttcaa ttacaccatt ttttagcagc
     7021 accggattaa cataactttc caaaatgttg tacgaaccgt taaacaaaaa cagttcacct
     7081 cccttttcta tactattgtc tgcgagcagt tgtttgttgt taaaaataac agccattgta
     7141 atgagacgca caaactaata tcaca
//
