LOCUS       pFB-LIC-Bse.        6816 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Baculovirus vector encoding an N-terminal 6xHis-TEV cassette and 
            ampicillin resistance plus a SacB negative selection marker, for 
            purification of recombinant proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pFB-LIC-Bse
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6816)
  AUTHORS   Savitsky P, Bray J, Cooper CD, Marsden BD, Mahajan P, Burgess-Brown 
            NA, Gileadi O.
  TITLE     High-throughput production of human proteins for crystallization: 
            the SGC experience.
  JOURNAL   J. Struct. Biol. 2010;172:3-13.
  PUBMED    20541610
REFERENCE   2  (bases 1 to 6816)
  AUTHORS   Structural Genomics Consortium
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6816)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct. 
            Biol."; date: "2010"; volume: "172"; pages: "3-13"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     For ligation-independent cloning (LIC), linearize with BseRI to 
            remove the SacB cassette, and treat with T4 DNA polymerase plus 
            dGTP.
FEATURES             Location/Qualifiers
     source          1..6816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..92
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     CDS             148..150
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             154..171
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             196..216
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
     protein_bind    complement(246..279)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        294..739
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             740..2158
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
     polyA_signal    2403..2537
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     mobile_element  complement(2566..2731)
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      2915..3370
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3397..3501
                     /label=AmpR promoter
     CDS             3502..4359
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      4533..5121
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     mobile_element  complement(5424..5648)
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(5718..6248)
                     /label=GmR
                     /note="gentamycin acetyltransferase"
     promoter        complement(6437..6465)
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc
       61 gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca
      121 tcgggcgcgg atctcggtcc gaaaaccatg ggccaccatc atcatcatca ttcttctggt
      181 gtagatctgg gtaccgagaa cctgtacttc caatccataa gctagcttct cctcctgaaa
      241 gatccataac ttcgtatagc atacattata cgaagttatg cggccgcgac gtccacatat
      301 acctgccgtt cactattatt tagtgaaatg agatattatg atattttctg aattgtgatt
      361 aaaaaggcaa ctttatgccc atgcaacaga aactataaaa aatacagaga atgaaaagaa
      421 acagatagat tttttagttc tttaggcccg tagtctgcaa atccttttat gattttctat
      481 caaacaaaag aggaaaatag accagttgca atccaaacga gagtctaata gaatgaggtc
      541 gaaaagtaaa tcgcgcgggt ttgttactga taaagcaggc aagacctaaa atgtgtaaag
      601 ggcaaagtgt atactttggc gtcacccctt acatatttta ggtctttttt tattgtgcgt
      661 aactaacttg ccatcttcaa acaggagggc tggaagaagc agaccgctaa cacagtacat
      721 aaaaaaggag acatgaacga tgaacatcaa aaagtttgca aaacaagcaa cagtattaac
      781 ctttactacc gcactgctgg caggaggcgc aactcaagcg tttgcgaaag aaacgaacca
      841 aaagccatat aaggaaacat acggcatttc ccatattaca cgccatgata tgctgcaaat
      901 ccctgaacag caaaaaaatg aaaaatataa agttcctgag ttcgattcgt ccacaattaa
      961 aaatatctct tctgcaaaag gcctggacgt ttgggacagc tggccattac aaaacactga
     1021 cggcactgtc gcaaactatc acggctacca catcgtcttt gcattagccg gagatcctaa
     1081 aaatgcggat gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa cttctattga
     1141 cagctggaaa aacgctggcc gcgtctttaa agacagcgac aaattcgatg caaatgattc
     1201 tatcctaaaa gaccaaacac aagaatggtc aggttcagcc acatttacat ctgacggaaa
     1261 aatccgttta ttctacactg atttctccgg taaacattac ggcaaacaaa cactgacaac
     1321 tgcacaagtt aacgtatcag catcagacag ctctttgaac atcaacggtg tagaggatta
     1381 taaatcaatc tttgacggtg acggaaaaac gtatcaaaat gtacagcagt tcatcgatga
     1441 aggcaactac agctcaggcg acaaccatac gctgagagat cctcactacg tagaagataa
     1501 aggccacaaa tacttagtat ttgaagcaaa cactggaact gaagatggct accaaggcga
     1561 agaatcttta tttaacaaag catactatgg caaaagcaca tcattcttcc gtcaagaaag
     1621 tcaaaaactt ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg gcgctctcgg
     1681 tatgattgag ctaaacgatg attacacact gaaaaaagtg atgaaaccgc tgattgcatc
     1741 taacacagta acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg gcaaatggta
     1801 cctgttcact gactcccgcg gatcaaaaat gacgattgac ggcattacgt ctaacgatat
     1861 ttacatgctt ggttatgttt ctaattcttt aactggccca tacaagccgc tgaacaaaac
     1921 tggccttgtg ttaaaaatgg atcttgatcc taacgatgta acctttactt actcacactt
     1981 cgctgtacct caagcgaaag gaaacaatgt cgtgattaca agctatatga caaacagagg
     2041 attctacgca gacaaacaat caacgtttgc gcctagcttc ctgctgaaca tcaaaggcaa
     2101 gaaaacatct gttgtcaaag acagcatcct tgaacaagga caattaacag ttaacaaata
     2161 aaaacgcaaa agaaaatgcc gatatcctat tggcattgac gtcaggtggc acttttcgag
     2221 gagtttacta gtaagtaaag gtggatacgg atccgaattc gagctccgtc gacaagcttg
     2281 tcgagaagta ctagaggatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     2341 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     2401 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     2461 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     2521 cttatcatgt ctggatctga tcactgatat cgcctaggag atccgaacca gataagtgaa
     2581 atctagttcc aaactatttt gtcattttta attttcgtat tagcttacga cgctacaccc
     2641 agttcccatc tattttgtca ctcttcccta aataatcctt aaaaactcca tttccacccc
     2701 tcccagttcc caactatttt gtccgcccac agcggggcat ttttcttcct gttatgtttt
     2761 taatcaaaca tcctgccaac tccatgtgac aaaccgtcat cttcggctac tttttctctg
     2821 tcacagaatg aaaatttttc tgtcatctct tcgttattaa tgtttgtaat tgactgaata
     2881 tcaacgctta tttgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2941 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3001 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3061 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3121 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3181 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     3241 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3301 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3361 tttacaattt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     3421 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     3481 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     3541 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     3601 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     3661 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     3721 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     3781 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     3841 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     3901 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     3961 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     4021 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     4081 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     4141 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     4201 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     4261 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     4321 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     4381 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4441 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4501 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4561 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4621 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4681 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4741 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4801 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4861 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4921 gagcattgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4981 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5041 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5101 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5161 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5221 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     5281 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     5341 ggtatttcac accgcagacc agccgcgtaa cctggcaaaa tcggttacgg ttgagtaata
     5401 aatggatgcc ctgcgtaagc gggtgtgggc ggacaataaa gtcttaaact gaacaaaata
     5461 gatctaaact atgacaataa agtcttaaac tagacagaat agttgtaaac tgaaatcagt
     5521 ccagttatgc tgtgaaaaag catactggac ttttgttatg gctaaagcaa actcttcatt
     5581 ttctgaagtg caaattgccc gtcgtattaa agaggggcgt ggccaagggc atggtaaaga
     5641 ctatattcgc ggcgttgtga caatttaccg aacaactccg cggccgggaa gccgatctcg
     5701 gcttgaacga attgttaggt ggcggtactt gggtcgatat caaagtgcat cacttcttcc
     5761 cgtatgccca actttgtata gagagccact gcgggatcgt caccgtaatc tgcttgcacg
     5821 tagatcacat aagcaccaag cgcgttggcc tcatgcttga gcagattgat gagcgcggtg
     5881 gcaatgccct gcctccggtg ctcgccggag actgcgagat catagatata gatctcacta
     5941 cgcggctgct caaacctggg cagaacgtaa gccgcgagag cgccaacaac cgcttcttgg
     6001 tcgaaggcag caagcgcgat gaatgtctta ctacggagca agttcccgag gtaatcggag
     6061 tccggctgat gttgggagta ggtggctacg tctccgaact cacgaccgaa aagatcaaga
     6121 gcagcccgca tggatttgac ttggtcaggg ccgagcctac atgtgcgaat gatgcccata
     6181 cttgagccac ctaactttgt tttagggcga ctgccctgct gcgtaacatc gttgctgctg
     6241 cgtaacatcg ttgctgctcc ataacatcaa acatcgaccc acggcgtaac gcgcttgctg
     6301 cttggatgcc cgaggcatag actgtacaaa aaaacagtca taacaagcca tgaaaaccgc
     6361 cactgcgccg ttaccaccgc tgcgttcggt caaggttctg gaccagttgc gtgagcgcat
     6421 acgctacttg cattacagtt tacgaaccga acaggcttat gtcaactggg ttcgtgcctt
     6481 catccgtttc cacggtgtgc gtcacccggc aaccttgggc agcagcgaag tcgaggcatt
     6541 tctgtcctgg ctggcgaacg agcgcaaggt ttcggtctcc acgcatcgtc aggcattggc
     6601 ggccttgctg ttcttctacg gcaaggtgct gtgcacggat ctgccctggc ttcaggagat
     6661 cggaagacct cggccgtcgc ggcgcttgcc ggtggtgctg accccggatg aagtggttcg
     6721 catcctcggt tttctggaag gcgagcatcg tttgttcgcc caggactcta gctatagttc
     6781 tagtggttgg ctacgtatac tccggaatat taatag
//
