LOCUS       sequence-224680-        7315 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 N-terminal monomeric GFP, cleavable with TEV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7315)
  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 7315)
  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: pOCC175
FEATURES             Location/Qualifiers
     source          1..7315
                     /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             230..247
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             260..1360
                     /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     1334..1357
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1415..1438
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1439..1441,1442..1444,1445..2155)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1484..1505)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1745..1764)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2092..2113
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2174..2194
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2303..2608
                     /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     2487..2506
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     polyA_signal    2781..2915
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2818..2837)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2872..2891
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           2988..3360
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(3712..3729)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3822..4410)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3902..3921)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4581..5372,5373..5441))
                     /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     5204..5223
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5443..5547)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5848..5870
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5862..5879
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5863..5879
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5890..5909
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5890..5908
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6519..6643
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6681..7286)
                     /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 ggcagcagcc atcaccatca
      241 ccatcacagc agcggcagaa tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg
      301 cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat
      361 taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac
      421 tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc
      481 tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac
      541 ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt
      601 atcgctgatt tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc
      661 ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga
      721 accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa
      781 cggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac
      841 cttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc
      901 agaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc
      961 caacatcgac accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca
     1021 accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa
     1081 agagctggca aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt
     1141 taataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggtgaa
     1201 agatccgcgt attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat
     1261 cccgcagatg tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg
     1321 tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cgaacaacaa
     1381 caacaataac aataacaaca acagcagcgg cagactggaa gttctgttcc aggggccgat
     1441 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1501 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     1561 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     1621 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     1681 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     1741 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1801 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     1861 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     1921 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     1981 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2041 cctgagcacc cagtccaaac tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2101 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggatc
     2161 cagcagcggc agagaaaact tgtatttcca gggcgcggcc gcaagcgcta tggccacaac
     2221 ctgttacatt gcacaagata aaaatatatc atcacgaaca gtaaaactgt ctgcttacat
     2281 aaacagtaat acaaggggtg ttatgcagtt taaggtttac acctataaaa gagagagccg
     2341 ttatcgtctg tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt
     2401 gatccccctg gccagtgccc gtctgctgtc agataaagtc tcccgtgaac tttacccggt
     2461 ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt
     2521 ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc
     2581 cattaacctg atgttctggg gaatataaat gtcaggctcc cttatacaca gccagtggcg
     2641 cgccgtaata gaagcttgtc gagaagtact agaggatcat aatcagccat accacatttg
     2701 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     2761 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     2821 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     2881 ccaaactcat caatgtatct tatcatgtct ggatctgatc actgcttgag cctaggagat
     2941 ccgaaccaga taagtgaaag cctgcatccg cagatcttaa ttaataaaac acgatacatt
     3001 gttattagta catttattaa gcgctagatt ctgtgcgttg ttgatttaca gacaattgtt
     3061 gtacgtattt taataattca ttaaatttat aatctttagg gtggtatgtt agagcgaaaa
     3121 tcaaatgatt ttcagcgtct ttatatctga atttaaatat taaatcctca atagatttgt
     3181 aaaataggtt tcgattagtt tcaaacaagg gttgtttttc cgaaccgatg gctggactat
     3241 ctaatggatt ttcgctcaac gccacaaaac ttgccaaatc ttgtagcagc aatctagctt
     3301 tgtcgatatt cgtttgtgtt ttgttttgta ataaaggttc gacgtcgttc aaaatattat
     3361 gcgcttttgt atttctttca tcactgtcgt tagtgtacaa ttgactcgac gtaaacacgt
     3421 taaataacgc ttggacatat ttaacatcgg gcgtgttagc tttattaggc cgattatcgt
     3481 cgtcgtccca accctcgtcg ttagaagttg cttccgaaga cgattttgcc atagccacac
     3541 gacgcctatt aattgtgtcg gctaacacgt ccgcgatcaa atttgtagtt gagctttttg
     3601 ggcctcatgg gccttccgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3661 gcattaacat ggtcatagct gtttccttgc gtattgggcg ctctccgctt cctcgctcac
     3721 tgactcgctg cgctcggtcg ttcgggtaaa gcctggggtg cctaatgagc aaaaggccag
     3781 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3841 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3901 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3961 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4021 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4081 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4141 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4201 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4261 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4321 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4381 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4441 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4501 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4561 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4621 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4681 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4741 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4801 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4861 ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg
     4921 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4981 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5041 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5101 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5161 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat
     5221 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5281 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5341 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5401 aaaaagggaa taagggcgac acggaaatgt tgaatactca ttactcttcc tttttcaata
     5461 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5521 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     5581 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     5641 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     5701 agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt gggaagggcg
     5761 tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     5821 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     5881 agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca aggccgtgac
     5941 gttaaaacta ttaagccatc caatcgaccg ttagtcgaat caggaccgct ggtgcgagaa
     6001 gccgcgaagt atggcgaatg catcgtataa cgtgtggagt ccgctcatta gagcgtcatg
     6061 tttagacaag aaagctacat atttaattga tcccgatgat tttattgata aattgaccct
     6121 aactccatac acggtattct acaatggcgg ggttttggtc aaaatttccg gactgcgatt
     6181 gtacatgctg ttaacggctc cgcccactat taatgaaatt aaaaattcca attttaaaaa
     6241 acgcagcaag agaaacattt gtatgaaaga atgcgtagaa ggaaagaaaa atgtcgtgga
     6301 catgctgaac aacaagatta atatgcctcc gtgtataaaa aaaatattga acgatttgaa
     6361 agaaaacaat gtaccgcgcg gcggtatgta caggaagagg tttatactaa actgttacat
     6421 tgcaaacgtg gtttcgtgtg ccaagtgtga aaaccgatgt ttaatcaagg ctctgacgca
     6481 tttctacaac cacgactcca agtgtgtggg tgaagtcatg catcttttaa tcaaatccca
     6541 agatgtgtat aaaccaccaa actgccaaaa aatgaaaact gtggacaagc tctgtccgtt
     6601 tgctggcaac tgcaagggtc tcaatcctat ttgtaattat tgaataataa aacaattata
     6661 aatgctaaat ttgtttttta ttaacgatac aaaccaaacg caacaagaac atttgtagta
     6721 ttatctataa ttgaaaacgc gtagttataa tcgctgaggt aatatttaaa atcattttca
     6781 aatgattcac agttaatttg cgacaatata attttatttt cacataaact agacgccttg
     6841 tcgtcttctt cttcgtattc cttctctttt tcatttttct cctcataaaa attaacatag
     6901 ttattatcgt atccatatat gtatctatcg tatagagtaa attttttgtt gtcataaata
     6961 tatatgtctt ttttaatggg gtgtatagta ccgctgcgca tagtttttct gtaatttaca
     7021 acagtgctat tttctggtag ttcttcggag tgtgttgctt taattattaa atttatataa
     7081 tcaatgaatt tgggatcgtc ggttttgtac aatatgttgc cggcatagta cgcagcttct
     7141 tctagttcaa ttacaccatt ttttagcagc accggattaa cataactttc caaaatgttg
     7201 tacgaaccgt taaacaaaaa cagttcacct cccttttcta tactattgtc tgcgagcagt
     7261 tgtttgttgt taaaaataac agccattgta atgagacgca caaactaata tcaca
//
