LOCUS       sequence-232369-        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        1..92
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     17..34
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     regulatory      134..143
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(146..151,152..175,176..211,212..235)
                     /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             248..1348
                     /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     1322..1345
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1400..1420
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1529..1834
                     /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     1713..1732
                     /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             1902..1919
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     polyA_signal    2061..2195
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2098..2117)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2152..2171
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     3'UTR           2268..2640
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     primer_bind     complement(2992..3009)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3102..3690)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3182..3201)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3861..4652,4653..4721))
                     /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     4484..4503
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4722..4826)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5127..5149
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5141..5158
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5142..5158
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5169..5188
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5169..5187
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5798..5922
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(5960..6565)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
ORIGIN
        1 atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc
       61 gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca
      121 tcgggcgcgg atcgccacca tgggcagcgc ttggagccat ccgcagtttg aaaaaggtgg
      181 tggtagcggt ggtggttcag gtggtagtgc atggtcacac cctcagtttg agaaaagcag
      241 cggcagaatg aaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta
      301 taacggtctc gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt
      361 tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc
      421 tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc
      481 tgaaatcacc ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt
      541 acgttacaac ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta
      601 taacaaagat ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa
      661 agaactgaaa gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac
      721 ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga
      781 cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga
      841 cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt
      901 taataaaggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac
      961 cagcaaagtg aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc
     1021 gttcgttggc gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcaaa
     1081 agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa
     1141 accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggtgaaag atccgcgtat
     1201 tgccgccact atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc
     1261 cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt
     1321 cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg aacaacaaca acaataacaa
     1381 taacaacaac agcagcggcg agaacctata cttccagtct gcggccgcaa gcgctatggc
     1441 cacaacctgt tacattgcac aagataaaaa tatatcatca cgaacagtaa aactgtctgc
     1501 ttacataaac agtaatacaa ggggtgttat gcagtttaag gtttacacct ataaaagaga
     1561 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg
     1621 gatggtgatc cccctggcca gtgcccgtct gctgtcagat aaagtctccc gtgaacttta
     1681 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt
     1741 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa
     1801 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggctccctta tacacagcca
     1861 gtggcgcgcc ggagaaccta tacttccagg gaggcagcag ccatcaccat caccatcaca
     1921 gtggataata gaagcttgtc gagaagtact agaggatcat aatcagccat accacatttg
     1981 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     2041 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     2101 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     2161 ccaaactcat caatgtatct tatcatgtct ggatctgatc actgcttgag cctaggagat
     2221 ccgaaccaga taagtgaaag cctgcatccg cagatcttaa ttaataaaac acgatacatt
     2281 gttattagta catttattaa gcgctagatt ctgtgcgttg ttgatttaca gacaattgtt
     2341 gtacgtattt taataattca ttaaatttat aatctttagg gtggtatgtt agagcgaaaa
     2401 tcaaatgatt ttcagcgtct ttatatctga atttaaatat taaatcctca atagatttgt
     2461 aaaataggtt tcgattagtt tcaaacaagg gttgtttttc cgaaccgatg gctggactat
     2521 ctaatggatt ttcgctcaac gccacaaaac ttgccaaatc ttgtagcagc aatctagctt
     2581 tgtcgatatt cgtttgtgtt ttgttttgta ataaaggttc gacgtcgttc aaaatattat
     2641 gcgcttttgt atttctttca tcactgtcgt tagtgtacaa ttgactcgac gtaaacacgt
     2701 taaataacgc ttggacatat ttaacatcgg gcgtgttagc tttattaggc cgattatcgt
     2761 cgtcgtccca accctcgtcg ttagaagttg cttccgaaga cgattttgcc atagccacac
     2821 gacgcctatt aattgtgtcg gctaacacgt ccgcgatcaa atttgtagtt gagctttttg
     2881 ggcctcatgg gccttccgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     2941 gcattaacat ggtcatagct gtttccttgc gtattgggcg ctctccgctt cctcgctcac
     3001 tgactcgctg cgctcggtcg ttcgggtaaa gcctggggtg cctaatgagc aaaaggccag
     3061 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3121 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3181 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3241 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3301 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3361 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3421 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3481 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3541 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3601 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3661 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3721 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3781 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3841 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3901 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3961 gagggcttac catctggccc cagtgctgca atgataccgc gagaaccacg ctcaccggct
     4021 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4081 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4141 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4201 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4261 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4321 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4381 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4441 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4501 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4561 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4621 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4681 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4741 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4801 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     4861 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4921 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4981 gtggccgcta cagggcgctc ccattcgcca ttcaggctgc gcaactgttg ggaagggcgt
     5041 ttcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     5101 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
     5161 gcgcgacgta atacgactca ctatagggcg aattggcgga aggccgtcaa ggccgtgacg
     5221 ttaaaactat taagccatcc aatcgaccgt tagtcgaatc aggaccgctg gtgcgagaag
     5281 ccgcgaagta tggcgaatgc atcgtataac gtgtggagtc cgctcattag agcgtcatgt
     5341 ttagacaaga aagctacata tttaattgat cccgatgatt ttattgataa attgacccta
     5401 actccataca cggtattcta caatggcggg gttttggtca aaatttccgg actgcgattg
     5461 tacatgctgt taacggctcc gcccactatt aatgaaatta aaaattccaa ttttaaaaaa
     5521 cgcagcaaga gaaacatttg tatgaaagaa tgcgtagaag gaaagaaaaa tgtcgtggac
     5581 atgctgaaca acaagattaa tatgcctccg tgtataaaaa aaatattgaa cgatttgaaa
     5641 gaaaacaatg taccgcgcgg cggtatgtac aggaagaggt ttatactaaa ctgttacatt
     5701 gcaaacgtgg tttcgtgtgc caagtgtgaa aaccgatgtt taatcaaggc tctgacgcat
     5761 ttctacaacc acgactccaa gtgtgtgggt gaagtcatgc atcttttaat caaatcccaa
     5821 gatgtgtata aaccaccaaa ctgccaaaaa atgaaaactg tggacaagct ctgtccgttt
     5881 gctggcaact gcaagggtct caatcctatt tgtaattatt gaataataaa acaattataa
     5941 atgctaaatt tgttttttat taacgataca aaccaaacgc aacaagaaca tttgtagtat
     6001 tatctataat tgaaaacgcg tagttataat cgctgaggta atatttaaaa tcattttcaa
     6061 atgattcaca gttaatttgc gacaatataa ttttattttc acataaacta gacgccttgt
     6121 cgtcttcttc ttcgtattcc ttctcttttt catttttctc ctcataaaaa ttaacatagt
     6181 tattatcgta tccatatatg tatctatcgt atagagtaaa ttttttgttg tcataaatat
     6241 atatgtcttt tttaatgggg tgtatagtac cgctgcgcat agtttttctg taatttacaa
     6301 cagtgctatt ttctggtagt tcttcggagt gtgttgcttt aattattaaa tttatataat
     6361 caatgaattt gggatcgtcg gttttgtaca atatgttgcc ggcatagtac gcagcttctt
     6421 ctagttcaat tacaccattt tttagcagca ccggattaac ataactttcc aaaatgttgt
     6481 acgaaccgtt aaacaaaaac agttcacctc ccttttctat actattgtct gcgagcagtt
     6541 gtttgttgtt aaaaataaca gccattgtaa tgagacgcac aaactaatat cacaagctgc
     6601 caccattttg ttcgcccagg actctagcta tagttctagt ggttggctac gtatactccg
     6661 gaatattaat ag
//
