LOCUS       pET28a(+)v2_TwinStrep-SUMO-FLP-8xHis        7081 bp    DNA     circular SYN 05-SEP-2025
DEFINITION  Expresses E. coli codon-optimized TwinStrep-SUMO-tagged FLP
            recombinase with N- and C-terminal NLS, using an improved pET
            design from Shilling et al. 2020.
ACCESSION
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
REFERENCE   1  (bases 1 to 6010)
  AUTHORS   Shilling PJ, Mirzadeh K, Cumming AJ, Widesheim M, Kock Z and Daley
            DO
  TITLE     Improved designs for pET expression plasmids increase protein
            production yield in Escherichia coli.
  JOURNAL   Commun Biol. 2020 May 7;3(1):214. doi: 10.1038/s42003-020-0939-8.
  PUBMED    32382055
REFERENCE   2  (bases 1 to 6010)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1
             type: "Journal Article"
             journalName: "Commun     Biol."
             date: "2020-05-7"
             volume: "3(1):214. doi"
             pages: "        10.1038/s42003-020-0939-8"                        
                             Sequence Label: pET28a T7pCONS TIR-2 sfGFP.
FEATURES             Location/Qualifiers
     rep_origin      12..467
                     /direction="RIGHT"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /Source_Sequence="referenceToDocument:
                     urn:local:.:4k6-jj3zxcq@7.2#0:[11,467)#5A1D76849B0FD863FA1
                     77BADD3624C53 label: pET28a(+)v2 PCR Product -
                     EcCO_TwinStrep-SUMO_PhiC_8xHis: 12->467"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="f1 ori"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:gj-jm1kter"">f1 ori</a>"
                     /label="f1 ori"
     CDS             complement(559..1374)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /note="confers resistance to kanamycin in bacteria or G418
                      (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLY
                     GKPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIP
                     GKTAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGL
                     VDASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVG
                     RVGIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
                     /label="KanR"
     rep_origin      1496..2084
                     /direction="RIGHT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /Source_Sequence="referenceToDocument:
                     urn:local:.:4k6-jj3zxcq@7.2#0:[1495,2084)#B9D487880B8951C1
                     E4053FFD589054A9 label: pET28a(+)v2 PCR Product -
                     EcCO_TwinStrep-SUMO_PhiC_8xHis: 1496->2084"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="ColE1 ori"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; ori (<a
                     href=""urn:local:.:gy-jm1kzmu"">ColE1 ori</a>)"
                     /label="ori"
     misc_feature    2270..2409
                     /note="basis of mobility region from pBR322"
                     /label="bom"
     CDS             complement(2511..2702)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low
                     copy number"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDH
                     ADELYRSCLARFGDDGENL"
                     /label="rop"
     CDS             complement(3511..4593)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE
                     LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS
                     GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG
                     DWSAMSGFQQTMQMLN*GIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT
                     EDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT
                     QTASPRALADSLMQLARQVSRLESGQ"
                     /label="lacI"
     promoter        complement(4594..4671)
                     /gene="lacI"
     promoter        4980..4998
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     misc_difference 4980..5002
                     /created_by="yl"
                     /Source_Sequence="referenceToDocument:
                     urn:local:.:ao-jgsfbkq@7.3#0:[4979,5002)#9FE6A36AFF5BBC927
                     C202C18B9EFF6AE label: pET28a(+)v2: 4980->5002"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="pET28a(+)v2 - Updated consensus T7
                     promoter sequence"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; Updated
                     consensus T7 promoter sequence (<a
                     href=""urn:local:.:ki-jj1upy0"">pET28a(+)v2 - Updated
                     consensus T7 promoter sequence</a>)"
                     /label="Updated consensus T7 promoter sequence"
     protein_bind    5003..5027
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /label="lac operator"
     RBS             5042..5064
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     misc_difference 5042..5070
                     /created_by="yl"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="RBS with optimized Shine-Dalgarno
                     region of TIR-2 for PET28a(+) translation"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:h2-jj1sf1f"">RBS with optimized
                     Shine-Dalgarno region of TIR-2 for PET28a(+)
                     translation</a>"
                     /label="RBS with optimized Shine-Dalgarno region of TIR-2
                     for PET28a(+) translation"
     misc_difference 5071..5079
                     /created_by="yl"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on protein similarity"
                     /Transferred_From="Optimized CDS region of TIR-2 for
                     PET28a(+) translation"
                     /Transferred_Translation_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0;
                     Optimized CDS region of TIR-2 for PET28a(+) (<a
                     href=""urn:local:.:9g-jixcg0l"">Optimized CDS region of
                     TIR-2 for PET28a(+) translation</a>)"
                     /label="Optimized CDS region of TIR-2 for PET28a(+)"
     ORF             5071..6942
                     /Genetic_code="Standard"
                     /Reading_frame=1
                     /created_by="ORF Finder"
                     /label="ORF 1 (frame 1)"
     misc_difference 5086..5175
                     /created_by="yl"
                     /modified_by="yl"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on protein similarity"
                     /Transferred_From="TwinStrep Tag translation"
                     /Transferred_Translation_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0;
                     TwinStrep Tag (<a
                     href=""urn:local:.:7e-jixbv5c"">TwinStrep Tag
                     translation</a>)"
                     /label="TwinStrep Tag"
     misc_difference 5176..5211
                     /created_by="yl"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on protein similarity"
                     /Transferred_From="3xGSSG translation"
                     /Transferred_Translation_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; 3X GSSG
                     Linker (<a href=""urn:local:.:15o-jhs9lq7"">3xGSSG
                     translation</a>)"
                     /label="3X GSSG Linker"
     CDS             5215..5505
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /Transferred_Translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSS
                     EIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEA
                     HREQIGG"
                     /Source_Sequence="referenceToDocument:
                     urn:local:.:5n-jg7aaew@2.0#0:[6487,6778)#3ACBE9AFE2EAD3D35
                     41666B1D40E3777 label: sequence-262117-: 6488->6778"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on protein similarity"
                     /Transferred_From="SUMO translation"
                     /Transferred_Translation_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; SUMO (<a
                     href=""urn:local:.:ao-jixd5m0"">SUMO translation</a>)"
                     /label="SUMO"
     misc_signal     5506..5526
                     /created_by="yl"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on protein similarity"
                     /Transferred_From="SV40 NLS translation"
                     /Transferred_Translation_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; SV40 NLS
                     (<a href=""urn:local:.:99-jixcfan"">SV40 NLS
                     translation</a>)"
                     /label="SV40 NLS"
     misc_difference 5527..6795
                     /created_by="yl"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on protein similarity"
                     /Transferred_From="Flippase translation"
                     /Transferred_Translation_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; FLP1
                     Recombinase (<a href=""urn:local:.:dd-jixed22"">Flippase
                     translation</a>)"
                     /Color="#ff1a00"
                     /modified_by="yl"
                     /label="FLP1 Recombinase"
     misc_difference 6796..6831
                     /created_by="yl"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on protein similarity"
                     /Transferred_From="3xGSSG translation"
                     /Transferred_Translation_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; 3X GSSG
                     Linker (<a href=""urn:local:.:15o-jhs9lq7"">3xGSSG
                     translation</a>)"
                     /label="3X GSSG Linker"
     CDS             6832..6879
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /Source_Sequence="referenceToDocument:
                     urn:local:.:5j-jg7a7xh@6.0#0:[4191,4239)#6CBB20B8FE951A7C8
                     5B950538D744DD3 label: sequence-408673-: 4192->4239"
                     /Original_Translation="KRPAATKKAGQAKKKK"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on protein similarity"
                     /Transferred_From="nucleoplasmin NLS (optimized)
                     translation"
                     /Transferred_Translation_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0;
                     nucleoplasmin NLS (<a
                     href=""urn:local:.:9n-jixchmp"">nucleoplasmin NLS
                     (optimized) translation</a>)"
                     /label="nucleoplasmin NLS"
     misc_difference 6880..6915
                     /created_by="yl"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on protein similarity"
                     /Transferred_From="3xGSSG translation"
                     /Transferred_Translation_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; 3X GSSG
                     Linker (<a href=""urn:local:.:15o-jhs9lq7"">3xGSSG
                     translation</a>)"
                     /label="3X GSSG Linker"
     domain          6916..6939
                     /created_by="yl"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on protein similarity"
                     /Transferred_From="8xHis translation"
                     /Transferred_Translation_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; 8x His
                     (<a href=""urn:local:.:15h-jhs9lg5"">8xHis
                     translation</a>)"
                     /label="8x His"
     terminator      7009..7056
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
                     /Source_Sequence="referenceToDocument:
                     urn:local:.:4k6-jj3zxcq@7.2#0:[7554,7602)#F18967AA1336538F
                     E70608863F6FB9C1 label: pET28a(+)v2 PCR Product -
                     EcCO_TwinStrep-SUMO_PhiC_8xHis: 7555->7602"
                     /note="Derived using Geneious Prime 2025.2.1 'Annotate
                     from Database' based on nucleotide similarity"
                     /Transferred_From="T7 terminator"
                     /Transferred_Similarity="100.00%"
                     /Primary_Match="100.00%; Adjusted-Interval-0:0/0; <a
                     href=""urn:local:.:ge-jm1krio"">T7 terminator</a>"
                     /label="T7 terminator"
ORIGIN      
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
      481 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
      541 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
      601 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
      661 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
      721 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
      781 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
      841 gacttgctca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
      901 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
      961 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     1021 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     1081 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     1141 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     1201 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     1261 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     1321 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     1381 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     1441 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     1501 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     1561 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     1621 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     1681 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1741 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1801 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1861 ccgaactgag atacctacag cgtgagctat tagaaagcgc cacgcttccc gaagggagaa
     1921 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     1981 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2041 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     2101 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     2161 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     2221 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     2281 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     2341 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     2401 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     2461 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     2521 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     2581 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     2641 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     2701 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     2761 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     2821 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     2881 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     2941 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     3001 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     3061 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     3121 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     3181 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     3241 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     3301 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     3361 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     3421 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     3481 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3541 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3601 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     3661 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     3721 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     3781 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     3841 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct aattcagcat
     3901 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     3961 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     4021 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     4081 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     4141 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     4201 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     4261 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     4321 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     4381 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     4441 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     4501 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     4561 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     4621 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     4681 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     4741 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     4801 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     4861 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     4921 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     4981 aatacgactc actataggga gaggaattgt gagcggataa caattcccct ctagaaataa
     5041 ttttgtttaa ctttaagaag gagagcagct atgcagcttg ggagctcagc ttggtcacat
     5101 cctcaatttg agaagggtgg tgggtccggt ggtgggtccg gtggatccgc atggagtcat
     5161 cctcaatttg agaagggttc gtcgggcggg tctagtggcg ggtcttctgg catgagcgac
     5221 tcagaggtga atcaagaggc caagccagag gtcaagcctg aggtcaagcc tgagacgcat
     5281 ataaatctta aggtaagtga tggaagctcg gagattttct ttaagattaa gaagacgacg
     5341 cccctcagac ggttaatgga agcatttgcc aagcgtcaag ggaaggagat ggattcgtta
     5401 cgttttcttt atgatggcat cagaatacaa gccgaccaaa ctcccgagga tcttgatatg
     5461 gaagacaatg atattatcga ggctcatcga gagcaaatcg gtggtccaaa gaagaagcgg
     5521 aaggtgatgt cccaatttga tatcttatgt aagacgcccc ctaaggtgtt agtgcggcaa
     5581 tttgtcgagc gctttgagcg gccctcgggc gagaagatcg cctcatgtgc agccgagtta
     5641 acatatctct gctggatgat aacgcataat ggaacagcta ttaagagagc cacttttatg
     5701 tcctacaata ccatcatttc caattcattg tcgttcgata tcgtcaataa gtcccttcaa
     5761 ttcaagtaca agacacaaaa ggccacaata ctcgaggcaa gcttaaagaa gttaatccct
     5821 gcatgggagt tcacgattat accatataat ggtcaaaagc atcaaagcga tataacagat
     5881 atcgtaagct cattacaatt gcaatttgag agtagtgagg aagccgataa gggcaattca
     5941 cattcgaaga agatgttaaa ggctcttctc agcgagggtg agtcaatttg ggagataact
     6001 gagaagatat taaattcgtt tgagtatacg agccgcttta ctaagacgaa gacgttatac
     6061 caattcttgt ttcttgcaac ctttatcaat tgtggacgct tcagcgatat caagaatgta
     6121 gaccctaagt cgtttaagtt agtccaaaat aagtacttgg gcgtcataat tcaatgttta
     6181 gtaacggaga caaagacgag tgtatcacgt catatatact tcttcagcgc ccgtgggcgg
     6241 atagacccct tagtgtacct cgacgagttc ttacggaatt ccgagcctgt gcttaagcgt
     6301 gtgaatagaa cggggaatag ttcatcgaat aagcaagagt atcaattact taaggacaat
     6361 ctcgtacggt cgtataataa ggcccttaag aagaatgcac catatccaat atttgcaatt
     6421 aagaatggac ccaagagcca tataggtaga catttaatga ctagctttct tagtatgaag
     6481 ggcttaactg agcttactaa tgtggtgggc aattggagtg acaagcgagc tagcgccgtg
     6541 gcccgaacta catatactca tcaaataact gcaatccccg accattattt tgccctcgta
     6601 tcccgctatt acgcatatga cccaatttca aaggagatga ttgcactcaa ggatgagacg
     6661 aatccaatag aggagtggca acatattgag caattgaagg gaagtgccga gggctccatt
     6721 cgctaccctg cttggaatgg tataataagc caagaggtgt tggattactt atcatcgtat
     6781 ataaatagac gcataggtag ctccggtggc tcaagcggtg gatcttcggg caagcgacca
     6841 gctgctacta agaaggctgg acaagccaag aagaagaagg gttcgtctgg cggatcgtca
     6901 ggtgggtcat caggccatca tcatcatcat caccatcact aatgagatcc ggctgctaac
     6961 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     7021 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     7081 t
//
