LOCUS       050_BB1_23_eGFP      2655 bp    DNA     circular UNA 07-Jan-2015
DEFINITION  old name: BB1_linker_FS2_BsaI_FS3
FEATURES             Location/Qualifiers
     CDS             join(1954..2655,1..117)
                     /label=KanR
     Vector          join(1669..2655,1..947)
                     /origin="BB1_L_23_syn_BsaI_1 (1.966bp circular)Target
                     vector: from BsaI cut at 979^980 to BsaI cut at 947^948,
                     1.934bpOrientation: originalPlus strand 5':   no
                     changePlus strand 3':   no changeMinus strand 5':  no
                     changeMinus strand 3':  no change"
     oriT            163..778
                     /label="ColE1 origin, high copy number"
     Primer          805..822
                     /label=Seq_BB1_FW
     Cleavage_site   join(<1669..1687,932..>947)
                     /label="Linker FS2 FS3"
     Insertion       948..1665
                     /origin="GFP_ (FSJ1(BsaI)_GFP_Fw - FSJ2(BsaI)_GFP_REV))
                     (744bp linear)Fragment: from BsaI cut at 11^12 to BsaI
                     cut at 729^730, 718bpOrientation: originalPlus strand 5':
                     no changePlus strand 3':   no changeMinus strand 5':  no
                     changeMinus strand 3':  no change"
                     /label=Insert
     Insertion       948..1668
                     /origin="Fragment (CDS_BB1_GFP (FSJ1(BsaI)_GFP_Fw fs2 -
                     FSJ2(BsaI)_GFP_REV fs3)) (747bp linear)Fragment: from
                     BsaI cut at 11^12 to BsaI cut at 732^733,
                     721bpOrientation: originalPlus strand 5':   no changePlus
                     strand 3':   no changeMinus strand 5':  no changeMinus
                     strand 3':  no change"
                     /label=Insert
     Primer_binding_ <948..968
                     /note="Primer: gaCagGtctcCaATGGTGAGCAAGGGCGAGGA"
                     /note="Number of mismatches: 9"
                     /note="Number of other matches on pG1_GFP_CYC1: 0"
                     /note="Primer binding region: 965..996"
                     /label="FSJ1(BsaI)_GFP_Fw (mismatches: 9)"
     CDS             <948..>1665
                     /label="'GFP ORF'"
     PCR_fragment    join(<948..968,969..1649,complement(1650..>1669))
                     /note="Forward primer name: FSJ1(BsaI)_GFP_Fw"
                     /note="Forward primer: gaCagGtctcCaATGGTGAGCAAGGGCGAGGA"
                     /note="Forward primer misprimes: 9"
                     /note="Reverse primer name: FSJ2(BsaI)_GFP_REV"
                     /note="Reverse primer: GatcgGtCtcCtTTACTTGTACAGCTCGTCC"
                     /note="Reverse primer misprimes: 8"
                     /note="Fragment length: 744"
                     /label="Fragment (pG1_GFP_CYC1 (FSJ1(BsaI)_GFP_Fw - FSJ2(BsaI)_GFP_REV))"
     PCR_fragment    join(<948..968,969..1646,complement(1647..>1672))
                     /note="Forward primer name: FSJ1(BsaI)_GFP_Fw fs2"
                     /note="Forward primer: gaCaggtctcCcATGGTGAGCAAGGGCGAGGA"
                     /note="Forward primer misprimes: 10"
                     /note="Reverse primer name: FSJ2(BsaI)_GFP_REV fs3"
                     /note="Reverse primer:
                     gatCGGtctccaagcTTACTTGTACAGCTCGTCCATG"
                     /note="Reverse primer misprimes: 12"
                     /note="Fragment length: 747"
                     /label="Fragment (CDS_BB1_GFP (FSJ1(BsaI)_GFP_Fw fs2 - FSJ2(BsaI)_GFP_REV fs3))"
     Primer_binding_ complement(1650..>1669)
                     /note="Primer: GatcgGtCtcCtTTACTTGTACAGCTCGTCC"
                     /note="Number of mismatches: 8"
                     /note="Number of other hits on pG1_GFP_CYC1: 1"
                     /note="Primer binding region: complement(1678..1708)"
                     /label="FSJ2(BsaI)_GFP_REV (mismatches: 8)"
     Region          <1666..>1668
                     /label=mcs
     Region          <1666..>1668
                     /label=Insert
     CDS             <1666..>1668
                     /label=lacZ
     Vector          1666..>1668
                     /origin="CDS_BB1 (2.974bp circular)Target vector: from
                     BsaI cut at 2183^2184 to BsaI cut at 2153^2154,
                     2.944bpOrientation: originalPlus strand 5':   no
                     changePlus strand 3':   no changeMinus strand 5':  no
                     changeMinus strand 3':  no change"
     FS              1669..1672
                     /label=FS3
     Primer          complement(1755..1771)
                     /label=Seq_BB1_BW
ORIGIN
        1 TGCCTCGGTG AGTTTTCTCC TTCATTACAG AAACGGCTTT TTCAAAAATA TGGTATTGAT
       61 AATCCTGATA TGAATAAATT GCAGTTTCAC TTGATGCTCG ATGAGTTTTT CTAATGAGGA
      121 CCTAAATGTA ATCACCTGGC TCACCTTCGG GTGGGCCTTT CTGCGTTGCT GGCGTTTTTC
      181 CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAT GCTCAAGTCA GAGGTGGCGA
      241 AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT
      301 CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG
      361 GCGCTTTCTC ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG
      421 CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT
      481 CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC
      541 AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC
      601 TACGGCTACA CTAGAAGAAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTCG
      661 GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT
      721 TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATTT
      781 TCTACCGAAG AAAGGCCCAC CCGTGTAAAA CGACGGCCAG TTTATCTAGT CAGCTTGATT
      841 CTAGCTGATC GTGGACCGGA AGGTGAGCCA GTGAGTTGAT TGCAGTCCAG TTACGCTGGA
      901 GTCTGAGGCT CGTCCTGAAT GATATGCGGC CTCGAATTCG AAGACGCCAT GGTGAGCAAG
      961 GGCGAGGAGC TGTTCACCGG GGTGGTGCCC ATCCTGGTCG AGCTGGACGG CGACGTAAAC
     1021 GGCCACAAGT TCAGCGTGTC CGGCGAGGGC GAGGGCGATG CCACCTACGG CAAGCTGACC
     1081 CTGAAGTTCA TCTGCACCAC CGGCAAGCTG CCCGTGCCCT GGCCCACCCT CGTGACCACC
     1141 CTGACCTACG GCGTGCAGTG CTTCAGCCGC TACCCCGACC ACATGAAGCA GCACGACTTC
     1201 TTCAAGTCCG CCATGCCCGA AGGCTACGTC CAGGAGCGCA CCATCTTCTT CAAGGACGAC
     1261 GGCAACTACA AGACCCGCGC CGAGGTGAAG TTCGAGGGCG ACACCCTGGT GAACCGCATC
     1321 GAGCTGAAGG GCATCGACTT CAAGGAGGAC GGCAACATCC TGGGGCACAA GCTGGAGTAC
     1381 AACTACAACA GCCACAACGT CTATATCATG GCCGACAAGC AGAAGAACGG CATCAAGGTG
     1441 AACTTCAAGA TCCGCCACAA CATCGAGGAC GGCAGCGTGC AGCTCGCCGA CCACTACCAG
     1501 CAGAACACCC CCATCGGCGA CGGCCCCGTG CTGCTGCCCG ACAACCACTA CCTGAGCACC
     1561 CAGTCCGCCC TGAGCAAAGA CCCCAACGAG AAGCGCGATC ACATGGTCCT GCTGGAGTTC
     1621 GTGACCGCCG CCGGGATCAC TCTCGGCATG GACGAGCTGT ACAAGTAAGC TTGCGTCTTC
     1681 GAATTCGCGC GTGATCTTAC GGCATTATAC GTATGATCGG TCCACGATCA GCTAGATTAT
     1741 CTAGTCAGCT TGATGTCATA GCTGTTTCCT GAGGCTCAAT ACTGACCATT TAAATCATAC
     1801 CTGACCTCCA TAGCAGAAAG TCAAAAGCCT CCGACCGGAG GCTTTTGACT TGATCGGCAC
     1861 GTAAGAGGTT CCAACTTTCA CCATAATGAA ATAAGATCAC TACCGGGCGT ATTTTTTGAG
     1921 TTATCGAGAT TTTCAGGAGC TAAGGAAGCT AAAATGAGCC ATATTCAACG GGAAACGTCT
     1981 TGCTTGAAGC CGCGATTAAA TTCCAACATG GATGCTGATT TATATGGGTA TAAATGGGCT
     2041 CGCGATAATG TCGGGCAATC AGGTGCGACA ATCTATCGAT TGTATGGGAA GCCCGATGCG
     2101 CCAGAGTTGT TTCTGAAACA TGGCAAAGGT AGCGTTGCCA ATGATGTTAC AGATGAGATG
     2161 GTCAGGCTAA ACTGGCTGAC GGAATTTATG CCTCTTCCGA CCATCAAGCA TTTTATCCGT
     2221 ACTCCTGATG ATGCATGGTT ACTCACCACT GCGATCCCAG GGAAAACAGC ATTCCAGGTA
     2281 TTAGAAGAAT ATCCTGATTC AGGTGAAAAT ATTGTTGATG CGCTGGCAGT GTTCCTGCGC
     2341 CGGTTGCATT CGATTCCTGT TTGTAATTGT CCTTTTAACG GCGATCGCGT ATTTCGTCTC
     2401 GCTCAGGCGC AATCACGAAT GAATAACGGT TTGGTTGGTG CGAGTGATTT TGATGACGAG
     2461 CGTAATGGCT GGCCTGTTGA ACAAGTCTGG AAAGAAATGC ATAAACTCTT GCCATTCTCA
     2521 CCGGATTCAG TCGTCACTCA TGGTGATTTC TCACTTGATA ACCTTATTTT TGACGAGGGG
     2581 AAATTAATAG GTTGTATTGA TGTTGGACGA GTCGGAATCG CAGACCGATA CCAGGATCTT
     2641 GCCATCCTAT GGAAC
//
