LOCUS       pSBK.134                4204 bp ds-DNA     circular SYN 04-JUN-2021
DEFINITION  aTc->A + Cho->B_ncRNA_ribozyme-free. pSol backbone.
ACCESSION
VERSION     .
KEYWORDS    pSOL His tsf.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
REFERENCE   1  (bases 1 to 3145)
  AUTHORS   Ron
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Oct 12, 2015 from SnapGene 2.8.2
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     misc_feature    1..66
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     primer_bind     1..17
                     /Sequence="CGCGTCCGAACCCAGCT"
                     /Hairpin_Tm="36.3"
                     /Tm="62.5"
                     /Self_Dimer_Tm="None"
                     /%GC="70.6"
                     /created_by="primer3"
                     /label="SBK_23"
     terminator      21..66
                     /created_by="santi.bhattaraikline"
                     /biology="LuxIA"
                     /iGEM_Part="BBa_B0011"
                     /direction="Bidirectional"
                     /modified_by="santi.bhattaraikline"
                     /label="LuxIA Terminator"
     primer_bind     29..86
                     /modified_by="santi.bhattaraikline"
                     /Mismatches=0
                     /%GC="34.5"
                     /Hairpin_Tm="57.9"
                     /Self_Dimer_Tm="40.9"
                     /Tm="66.0"
                     /annotation_group="1153508646253400460ab021-8bf6-4352-995d
                     -1cf573fd6f03"
                     /Sequence="TAAAAAGCCAGATTATTAATCCGGCTTTTTTATTATTTCGCTTAACG
                     ATCGTTGGCTG"
                     /created_by="primer3"
                     /label="dualProm_bb_fow"
     primer_bind     complement(65..86)
                     /Sequence="CAGCCAACGATCGTTAAGCGAA"
                     /Hairpin_Tm="53.5"
                     /Tm="61.8"
                     /Self_Dimer_Tm="32.3"
                     /%GC="50.0"
                     /created_by="primer3"
                     /label="SBK_24"
     source          join(165..>1408,<67..164)
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      67
                     /label="L3S3P21"
     misc_feature    68..1406
                     /Original_Bases="ACCACAATTCAGCAAATTGTGAACATCATCACGTTCATCTT
                     TCCCTGGTTGCCAATGGCCCATTTTCCTGTCAGTAACGAGAAGGTCGCGAATTCAGGC
                     GCTTTTTAGACTGGTCGTAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTT
                     TAACTATAAGAAGGAGATATACATATGCATCATCACCACCATCACATGGCAGAAATTA
                     CGGCTTCCCTGGTGAAAGAATTGCGCGAACGTACGGGTGCAGGCATGATGGACTGCAA
                     GAAGGCGCTGACCGAAGCCAATGGCGACATCGAGCTGGCGATCGAGAACATGCGTAAA
                     TCGGGTGCAATCAAAGCGGCCAAAAAGGCGGGTAATGTTGCGGCAGACGGCGTCATTA
                     AAACCAAGATCGATGGTAACTATGGCATTATTCTGGAAGTCAACTGTCAGACGGATTT
                     CGTGGCGAAGGACGCGGGTTTTCAAGCCTTCGCCGACAAGGTCCTGGACGCAGCGGTG
                     GCTGGTAAAATCACGGATGTCGAAGTGTTGAAAGCACAGTTTGAAGAAGAGCGTGTCG
                     CTCTGGTGGCAAAGATCGGCGAGAATATCAATATCCGCCGTGTTGCGGCACTGGAAGG
                     TGATGTTCTGGGCAGCTACCAGCACGGTGCGCGTATTGGCGTTCTGGTGGCTGCCAAG
                     GGTGCGGACGAAGAGCTGGTGAAACATATCGCGATGCATGTCGCCGCGAGCAAACCAG
                     AGTTTATCAAACCGGAAGATGTTAGCGCCGAGGTCGTGGAAAAAGAGTATCAGGTACA
                     GCTGGACATTGCGATGCAGTCCGGCAAACCTAAAGAGATCGCAGAGAAGATGGTCGAG
                     GGCCGCATGAAGAAGTTTACCGGTGAGGTTAGCCTGACTGGTCAACCGTTTGTTATGG
                     AGCCGAGCAAAACGGTTGGTCAATTGCTGAAAGAGCACAATGCGGAAGTGACCGGTTT
                     CATTCGTTTTGAGGTGGGTGAAGGCATTGAGAAAGTGGAAACCGATTTCGCGGCAGAA
                     GTTGCGGCTATGAGCAAGCAATCTGAGAATCTGTACTTCCAGGGTTAATAGAGCGGCC
                     GCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGG
                     GTTTTT"
                     /label="ACCACAATTCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTT
                     GCCAATGGCCCATTTTCCTGTCAGTAACGAGAAGGTCGCGAATTCAGGCGCTTTTTAG
                     ACTGGTCGTAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTATAA
                     GAAGGAGATATACATATGCATCATCACCACCATCACATGGCAGAAATTACGGCTTCCC
                     TGGTGAAAGAATTGCGCGAACGTACGGGTGCAGGCATGATGGACTGCAAGAAGGCGCT
                     GACCGAAGCCAATGGCGACATCGAGCTGGCGATCGAGAACATGCGTAAATCGGGTGCA
                     ATCAAAGCGGCCAAAAAGGCGGGTAATGTTGCGGCAGACGGCGTCATTAAAACCAAGA
                     TCGATGGTAACTATGGCATTATTCTGGAAGTCAACTGTCAGACGGATTTCGTGGCGAA
                     GGACGCGGGTTTTCAAGCCTTCGCCGACAAGGTCCTGGACGCAGCGGTGGCTGGTAAA
                     ATCACGGATGTCGAAGTGTTGAAAGCACAGTTTGAAGAAGAGCGTGTCGCTCTGGTGG
                     CAAAGATCGGCGAGAATATCAATATCCGCCGTGTTGCGGCACTGGAAGGTGATGTTCT
                     GGGCAGCTACCAGCACGGTGCGCGTATTGGCGTTCTGGTGGCTGCCAAGGGTGCGGAC
                     GAAGAGCTGGTGAAACATATCGCGATGCATGTCGCCGCGAGCAAACCAGAGTTTATCA
                     AACCGGAAGATGTTAGCGCCGAGGTCGTGGAAAAAGAGTATCAGGTACAGCTGGACAT
                     TGCGATGCAGTCCGGCAAACCTAAAGAGATCGCAGAGAAGATGGTCGAGGGCCGCATG
                     AAGAAGTTTACCGGTGAGGTTAGCCTGACTGGTCAACCGTTTGTTATGGAGCCGAGCA
                     AAACGGTTGGTCAATTGCTGAAAGAGCACAATGCGGAAGTGACCGGTTTCATTCGTTT
                     TGAGGTGGGTGAAGGCATTGAGAAAGTGGAAACCGATTTCGCGGCAGAAGTTGCGGCT
                     ATGAGCAAGCAATCTGAGAATCTGTACTTCCAGGGTTAATAGAGCGGCCGCCACCGCT
                     GAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTT"
                     /note="Geneious type: Editing History Replacement"
     promoter        87..162
                     /label="pTet*"
     operator        109..127
                     /label="TetO2"
     promoter        128..133
                     /label="-35"
     operator        134..152
                     /label="tetO2"
     promoter        151..156
                     /label="-10"
     misc_RNA        165..>185
                     /note="hammerhead ribozyme from the tobacco ringspot virus
                      satellite RNA (Khvorova et al., 2003)"
                     /label="sTRSV HHRz"
     primer_bind     complement(165..192)
                     /Mismatches=0
                     /%GC="50.0"
                     /Hairpin_Tm="51.3"
                     /Self_Dimer_Tm="None"
                     /Tm="65.5"
                     /annotation_group="1962258059374005d2d814c-deaf-4901-88a9-
                     68222a981b4e"
                     /Sequence="CTTATCAGGAAAGCACATCCGGTGACAG"
                     /created_by="primer3"
                     /label="pSBK_106_4kld_rev"
     primer_bind     complement(165..192)
                     /Mismatches=0
                     /%GC="50.0"
                     /Hairpin_Tm="51.3"
                     /Self_Dimer_Tm="None"
                     /Tm="65.5"
                     /annotation_group="284591409482100e7834f0e-c0fc-4571-9b97-
                     9839d7b2ecc9"
                     /Sequence="CTTATCAGGAAAGCACATCCGGTGACAG"
                     /created_by="primer3"
                     /label="106_4kld_rev"
     misc_signal     <186..199
                     /created_by="User"
                     /Transferred_From="template"
                     /Transferred_Similarity="100.00%"
                     /label="T7 to msr"
     primer_bind     193..219
                     /Mismatches=0
                     /%GC="55.6"
                     /Hairpin_Tm="57.4"
                     /Self_Dimer_Tm="14.9"
                     /Tm="68.4"
                     /annotation_group="19622560814630077c3535e-9501-495b-9d7d-
                     4e360002ed94"
                     /Sequence="ATTCCGTATGCGCACCCTTAGCGAGAG"
                     /created_by="primer3"
                     /label="pSBK_106_4kld_fow"
     primer_bind     193..219
                     /Mismatches=0
                     /%GC="55.6"
                     /Hairpin_Tm="57.4"
                     /Self_Dimer_Tm="14.9"
                     /Tm="68.4"
                     /annotation_group="284591890921200b872ac8f-b2ff-4c38-88c0-
                     88ef8e648fcc"
                     /Sequence="ATTCCGTATGCGCACCCTTAGCGAGAG"
                     /created_by="primer3"
                     /label="106_4kld_fow"
     misc_rna        200..281
                     /created_by="User"
                     /Transferred_From="template"
                     /Transferred_Similarity="100.00%"
                     /label="msdRNA"
     misc_feature    201..212
                     /created_by="User"
                     /Transferred_From="template"
                     /Transferred_Similarity="100.00%"
                     /label="'primer'"
     primer_bind     228..253
                     /Mismatches=0
                     /%GC="38.5"
                     /Hairpin_Tm="48.0"
                     /Self_Dimer_Tm="14.8"
                     /Tm="60.9"
                     /annotation_group="104675332340500b6b4c216-dd69-42a3-86af-
                     7cb84c005db4"
                     /Sequence="TTAAGGTCAACCTCTGGATGTTGTTT"
                     /created_by="primer3"
                     /label="Uloop_detect_fow_wt"
     motif           243..250
                     /created_by="santi.bhattaraikline"
                     /label="Stem (RT Recognition)"
     motif           251..253
                     /created_by="santi.bhattaraikline"
                     /label="UUU Loop (RT Recognition)"
     primer          complement(254..>270)
                     /created_by="User"
                     /label="86_rev"
     motif           complement(254..261)
                     /created_by="santi.bhattaraikline"
                     /label="Stem (RT Recognition)"
     primer          complement(260..>270)
                     /modified_by="User"
                     /label="msd_Rev"
     misc_feature    complement(271..327)
                     /created_by="User"
                     /modified_by="Seth"
                     /label="msd"
     motif           271..287
                     /created_by="santi.bhattaraikline"
                     /label="Flanking"
     primer_bind     complement(283..305)
                     /modified_by="santi.bhattaraikline"
                     /Mismatches=0
                     /%GC="47.8"
                     /Hairpin_Tm="38.3"
                     /Self_Dimer_Tm="30.1"
                     /Tm="60.9"
                     /annotation_group="104674207201700e7392e35-4c4f-4e4f-a911-
                     7d8f5aff4ae7"
                     /Sequence="GTTGCTGCAACCTCTCCATTTTC"
                     /created_by="primer3"
                     /label="SPCR_Ec86_rev"
     motif           288..298
                     /created_by="santi.bhattaraikline"
                     /label="Stem"
     motif           299..301
                     /created_by="santi.bhattaraikline"
                     /label="Loop"
     motif           complement(302..312)
                     /created_by="santi.bhattaraikline"
                     /label="Stem"
     motif           complement(313..327)
                     /created_by="santi.bhattaraikline"
                     /label="Flanking"
     primer_bind     complement(314..336)
                     /Mismatches=0
                     /Mismatches=0
                     /%GC="47.8"
                     /Hairpin_Tm="43.0"
                     /Self_Dimer_Tm="None"
                     /Tm="60.3"
                     /annotation_group="10467439459930065f96e8e-e864-43dd-9624-
                     fec679ccd6d7"
                     /Sequence="CACCCTTACGTCAGAAAAAACGG"
                     /created_by="primer3"
                     /label="020_4kld_rev"
     primer          <328..338
                     /created_by="User"
                     /label="86_fow"
     primer          <328..336
                     /modified_by="User"
                     /label="msd_Fow"
     misc_feature    329..355
                     /created_by="User"
                     /label="'primer'"
     primer_bind     complement(333..365)
                     /Mismatches=0
                     /%GC="51.5"
                     /Hairpin_Tm="36.7"
                     /Self_Dimer_Tm="14.9"
                     /Tm="70.6"
                     /annotation_group="10467476893350072e34779-e262-45a8-9e22-
                     09ffb8fb62b1"
                     /Sequence="TGGTACCGAGTGATAAGATTCCGTATGCGCACC"
                     /created_by="primer3"
                     /label="057_ins_rev"
     primer_bind     346..379
                     /Mismatches=0
                     /%GC="35.3"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="13.5"
                     /Tm="64.6"
                     /annotation_group="1046738310769001cc628ef-d56a-4904-a5bc-
                     f6e220dbdcf5"
                     /Sequence="AATCTTATCACTCGGTACCAAATTCCAGAAAAGA"
                     /created_by="primer3"
                     /label="057_bb_fow"
     terminator      356..416
                     /label="L3S2P21"
     promoter        421..474
                     /label="pLacI"
     promoter        439..444
                     /label="-35"
     primer_bind     complement(440..460)
                     /%GC="52.4"
                     /Mismatches=0
                     /Hairpin_Tm="57.9"
                     /Self_Dimer_Tm="24.1"
                     /Tm="61.0"
                     /annotation_group="284590865444800d786b970-062d-4750-b946-
                     4197cd54f1a9;
                     2845917708145001ecac8d4-9576-4169-86bf-434431c7c6c0"
                     /Sequence="CATACCGCGAAAGGTTTTGCG"
                     /created_by="primer3"
                     /label="SBK_19; SBK_19"
     promoter        462..467
                     /label="-10"
     RBS             475..502
                     /label="tet1"
     CDS             503..1126
                     /label="TetR"
     primer_bind     complement(775..794)
                     /modified_by="santi.bhattaraikline"
                     /Mismatches=0
                     /%GC="50.0"
                     /Hairpin_Tm="41.2"
                     /Self_Dimer_Tm="42.4"
                     /Tm="60.1"
                     /annotation_group="2856355356733006c8e639b-8492-4dfb-8fe1-
                     e4a4abfb3565"
                     /Sequence="TTGCTCCATCGCGATGACTT"
                     /created_by="primer3"
                     /label="SBK_21_TetR"
     terminator      1136..1388
                     /label="induction operon terminator region"
     primer_bind     complement(1315..1334)
                     /Mismatches=0
                     /%GC="55.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="19.2"
                     /Tm="58.4"
                     /annotation_group="1046760830386002bd9f3f5-f89e-4737-a897-
                     1220eead4e12"
                     /Sequence="GTGATGTCGGCGATATAGGC"
                     /created_by="primer3"
                     /label="SLS_138"
     primer_bind     complement(1389..1418)
                     /Mismatches=0
                     /%GC="56.7"
                     /Hairpin_Tm="51.5"
                     /Self_Dimer_Tm="2.1"
                     /Tm="71.2"
                     /annotation_group="10641735748460073cc2781-1920-49a3-92a1-
                     32d71b86d034"
                     /Sequence="CTCCTTTCAGCAGTGAGCGAGGAAGCACCT"
                     /created_by="primer3"
                     /label="081_ins_rev"
     primer_bind     complement(1389..1408)
                     /Mismatches=0
                     /%GC="60.0"
                     /Hairpin_Tm="53.9"
                     /Self_Dimer_Tm="2.1"
                     /Tm="62.2"
                     /annotation_group="1046751456451003f965686-49f9-4a42-913c-
                     1cbc5697e8c6"
                     /Sequence="CAGTGAGCGAGGAAGCACCT"
                     /created_by="primer3"
                     /label="mari_cassette_rev"
     primer_bind     complement(1389..1408)
                     /Mismatches=0
                     /%GC="60.0"
                     /Hairpin_Tm="53.9"
                     /Self_Dimer_Tm="2.1"
                     /Tm="62.2"
                     /annotation_group="2845901932881002df1bddf-1276-4998-aeeb-
                     2d6f04d92181"
                     /Sequence="CAGTGAGCGAGGAAGCACCT"
                     /created_by="primer3"
                     /label="mari_cassette_rev"
     primer_bind     1399..1431
                     /Mismatches=0
                     /%GC="51.5"
                     /Hairpin_Tm="58.9"
                     /Self_Dimer_Tm="None"
                     /Tm="70.1"
                     /annotation_group="106416126034400617976fb-02a7-48c7-bd48-
                     576d6ea44203"
                     /Sequence="TCGCTCACTGCTGAAAGGAGGAACTATATCCGG"
                     /created_by="primer3"
                     /label="081_bb_fow"
     primer_bind     1409..1431
                     /Sequence="CTGAAAGGAGGAACTATATCCGG"
                     /Hairpin_Tm="44.6"
                     /Tm="57.9"
                     /Self_Dimer_Tm="None"
                     /%GC="47.8"
                     /created_by="primer3"
                     /label="pSol_bb_fow"
     terminator      1506..1535
                     /note="T3Te terminator"
                     /note="phage T3 early transcription terminator"
     primer_bind     complement(1506..1523)
                     /%GC="66.7"
                     /Mismatches=0
                     /Hairpin_Tm="57.6"
                     /Self_Dimer_Tm="4.7"
                     /Tm="60.4"
                     /annotation_group="284591589608600b212e4d8-7dd0-4675-a5c3-
                     dacae357daf4;
                     284591830755500a6f89ee9-87cc-45d6-9d64-7dd0155b9a6e"
                     /Sequence="CACCCGTGAAGGTGAGCC"
                     /created_by="primer3"
                     /label="SBK_012_T3Te_term; T3Te_term_SBK_012"
     rep_origin      1557..2144
                     /direction="RIGHT"
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2106..2125
                     /Mismatches=0
                     /%GC="45.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="56.7"
                     /annotation_group="2845902589623002b5f1d38-7a25-46c9-9190-
                     649cc22ab91e"
                     /Sequence="TTTTTGTGATGCTCGTCAGG"
                     /created_by="primer3"
                     /label="SLS_152"
     terminator      2156..2183
                     /note="T7Te terminator"
                     /note="phage T7 early transcription terminator"
     CDS             complement(2207..2398)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low
                     copy number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDH
                     ADELYRSCLARFGDDGENL"
     primer_bind     complement(2375..2408)
                     /Sequence="CGGGGCGTAAATGACCAAACAGGAAAAAACCGCC"
                     /Hairpin_Tm="46.4"
                     /Tm="72.7"
                     /Self_Dimer_Tm="None"
                     /%GC="52.9"
                     /created_by="primer3"
                     /label="15chlor_bb_rev"
     primer_bind     2389..2414
                     /Sequence="GTTTGGTCATTTACGCCCCGCCCTGC"
                     /Hairpin_Tm="None"
                     /Tm="70.4"
                     /Self_Dimer_Tm="None"
                     /%GC="61.5"
                     /created_by="primer3"
                     /label="15chlor_ins_fow"
     CDS             complement(2399..3058)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAF
                     LKTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETF
                     SSLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNV
                     ANMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG
                     A"
                     /label="CmR"
     source          <2399..>3058
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    2402..3055
                     /Original_Bases="GAAAAACTCATCGAGCATCAAGTGAAACTGCAATTTATTCA
                     TATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAA
                     CTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACT
                     CGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTG
                     AGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTC
                     TTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCACCA
                     ACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCCGT
                     TAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGC
                     ATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTC
                     CCTGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGA
                     TGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGCCTGACCATCTCATCTGTAAC
                     ATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTC
                     CCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTAT
                     ACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTC
                     CCGTTGAATATGGCT"
                     /label="GAAAAACTCATCGAGCATCAAGTGAAACTGCAATTTATTCATATCAGGAT
                     TATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAG
                     GCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACA
                     TCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCAC
                     CATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGAC
                     TTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCACCAACCAAACCG
                     TTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCCGTTAAAAGGAC
                     AATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAAT
                     ATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCTGGGATC
                     GCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAA
                     GAGGCATAAATTCCGTCAGCCAGTTTAGCCTGACCATCTCATCTGTAACATCATTGGC
                     AACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAT
                     CGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATA
                     AATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAAT
                     ATGGCT"
                     /note="Geneious type: Editing History Replacement"
     primer_bind     2969..2989
                     /Sequence="TTGAGCAACTGACTGAAATGC"
                     /Hairpin_Tm="37.3"
                     /Tm="57.4"
                     /Self_Dimer_Tm="None"
                     /%GC="42.9"
                     /created_by="primer3"
                     /label="SBK_16_CmR"
     primer_bind     complement(3031..3068)
                     /Sequence="TTCAGGAGCTATGGAGAAAAAAATCACTGGATATACCA"
                     /Hairpin_Tm="45.7"
                     /Tm="60.5"
                     /Self_Dimer_Tm="None"
                     /%GC="36.8"
                     /created_by="primer3"
                     /label="15chlor_ins_rev"
     primer_bind     3049..3084
                     /Sequence="TTTTCTCCATAGCTCCTGAAAATCTCGATAACTCAA"
                     /Hairpin_Tm="31.3"
                     /Tm="65.8"
                     /Self_Dimer_Tm="None"
                     /%GC="36.1"
                     /created_by="primer3"
                     /label="15chlor_bb_fow"
     promoter        complement(3059..3149)
                     /note="cat promoter"
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     3123..3144
                     /%GC="50.0"
                     /Mismatches=0
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="60.8"
                     /annotation_group="2845908055434005f602c85-fe55-4a8c-adaf-
                     3486330e62f2;
                     284591650504400937ed903-2a19-4c20-bed4-6153426be9db"
                     /Sequence="AAGTTGGAACCTCTTACGTGCC"
                     /created_by="primer3"
                     /label="cat_promoter_rev_SBK_015; SBK_015_cat_prom_rev"
     terminator      3150..3181
                     /note="tonB terminator"
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     primer_bind     3155..3172
                     /%GC="66.7"
                     /Mismatches=0
                     /Hairpin_Tm="67.2"
                     /Self_Dimer_Tm="39.5"
                     /Tm="61.4"
                     /annotation_group="2845900712768004c301b1b-5878-4ab2-9d08-
                     b4ffc46ac95f;
                     284591469507400ed5e95f0-fd25-4f3a-aea5-8cb76c5c5c55"
                     /Sequence="AAAGCCTCCGGTCGGAGG"
                     /created_by="primer3"
                     /label="SBK_013_tonB_term; tonB_term_SBK_013"
     primer_bind     complement(3187..3223)
                     /Mismatches=0
                     /%GC="40.5"
                     /Hairpin_Tm="28.9"
                     /Self_Dimer_Tm="None"
                     /Tm="60.2"
                     /annotation_group="284590012173800897c8ffd-401f-4ba8-b031-
                     2279f00f962d"
                     /Sequence="GACCGATTGACATTTAAATCATACCTGACCTCCATAG"
                     /created_by="primer3"
                     /label="dualProm_bb_rev_ins118"
     primer_bind     complement(3187..3213)
                     /Mismatches=0
                     /%GC="37.0"
                     /Hairpin_Tm="33.2"
                     /Self_Dimer_Tm="None"
                     /Tm="57.8"
                     /annotation_group="28459201314150087708ca4-e8a8-4a75-a26b-
                     5e944874382d"
                     /Sequence="CATTTAAATCATACCTGACCTCCATAG"
                     /created_by="primer3"
                     /label="dualProm_bb_rev"
     primer_bind     3204..3233
                     /Mismatches=0
                     /%GC="43.3"
                     /Hairpin_Tm="41.7"
                     /Self_Dimer_Tm="5.2"
                     /Tm="66.0"
                     /annotation_group="115396463717470068520c6f-c737-4e57-abdb
                     -4f761656029b"
                     /Sequence="GATTTAAATGTCAATCGGTCGGCAGATGCT"
                     /created_by="primer3"
                     /label="frag118_ins_fow"
     misc_feature    3214..4204
                     /Original_Bases="GTCAGTATTGAGCGATATCTAGAGAATTCGTC"
                     /label="GTCAGTATTGAGCGATATCTAGAGAATTCGTC"
                     /note="Geneious type: Editing History Replacement"
     CDS             complement(3214..3801)
                     /label="BetI-AM"
     source          join(complement(<3214..4139),complement(4140..>4204))
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     mutation        complement(3457..3459)
                     /label="S115I"
     primer_bind     3521..3540
                     /modified_by="santi.bhattaraikline"
                     /Mismatches=0
                     /%GC="55.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="6.8"
                     /Tm="60.3"
                     /annotation_group="285635347920800e3583371-37f0-4fac-be6e-
                     05e68a4afda2"
                     /Sequence="CCACCAACAATTGCCTGCAG"
                     /created_by="primer3"
                     /label="SBK_20_BetI-AM"
     RBS             complement(3802..3829)
                     /label="bet2"
     promoter        complement(3830..3883)
                     /label="pLacIQ"
     promoter        complement(3837..3842)
                     /label="-10"
     promoter        complement(3860..3865)
                     /label="-35"
     terminator      complement(3888..3948)
                     /label="L3S2P21"
     primer_bind     complement(3925..3958)
                     /Mismatches=0
                     /%GC="35.3"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="13.5"
                     /Tm="64.6"
                     /annotation_group="1046790953804000554fb98-55b9-4aaf-9e3c-
                     485317b35129"
                     /Sequence="AATCTTATCACTCGGTACCAAATTCCAGAAAAGA"
                     /created_by="primer3"
                     /label="057_bb_fow"
     primer_bind     3939..3971
                     /Mismatches=0
                     /%GC="51.5"
                     /Hairpin_Tm="36.7"
                     /Self_Dimer_Tm="14.9"
                     /Tm="70.6"
                     /annotation_group="104680032470000eee03c8e-4c30-46c4-a320-
                     08f6a29a3ea3"
                     /Sequence="TGGTACCGAGTGATAAGATTCCGTATGCGCACC"
                     /created_by="primer3"
                     /label="057_ins_rev"
     misc_feature    3949..3975
                     /created_by="User"
                     /label="'primer'"
     primer          complement(3966..>3976)
                     /created_by="User"
                     /label="86_fow"
     primer          complement(3968..>3976)
                     /modified_by="User"
                     /label="msd_Fow"
     primer_bind     3968..3990
                     /Mismatches=0
                     /Mismatches=0
                     /%GC="47.8"
                     /Hairpin_Tm="43.0"
                     /Self_Dimer_Tm="None"
                     /Tm="60.3"
                     /annotation_group="104679656511300cbc35898-56bd-4688-9de6-
                     6c6324f4b17d"
                     /Sequence="CACCCTTACGTCAGAAAAAACGG"
                     /created_by="primer3"
                     /label="020_4kld_rev"
     misc_feature    3977..4033
                     /created_by="User"
                     /modified_by="Seth"
                     /label="msd"
     motif           3977..3991
                     /created_by="santi.bhattaraikline"
                     /label="Flanking"
     source          complement(<3992..>4016)
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     motif           3992..4001
                     /created_by="santi.bhattaraikline"
                     /label="Stem"
     motif           4002..4006
                     /created_by="santi.bhattaraikline"
                     /label="Loop"
     motif           complement(4007..4016)
                     /created_by="santi.bhattaraikline"
                     /label="Stem"
     motif           complement(4017..4033)
                     /created_by="santi.bhattaraikline"
                     /label="Flanking"
     misc_rna        complement(4023..4104)
                     /created_by="User"
                     /Transferred_From="template"
                     /Transferred_Similarity="100.00%"
                     /label="msdRNA"
     primer          <4034..4050
                     /created_by="User"
                     /label="86_rev"
     primer          <4034..4044
                     /modified_by="User"
                     /label="msd_Rev"
     motif           4043..4050
                     /created_by="santi.bhattaraikline"
                     /label="Stem (RT Recognition)"
     motif           4051..4053
                     /created_by="santi.bhattaraikline"
                     /label="UUU Loop (RT Recognition)"
     primer_bind     complement(4051..4076)
                     /Mismatches=0
                     /%GC="38.5"
                     /Hairpin_Tm="48.0"
                     /Self_Dimer_Tm="14.8"
                     /Tm="60.9"
                     /annotation_group="104680593630300661eb691-a662-481c-9ce9-
                     5b569244d39d"
                     /Sequence="TTAAGGTCAACCTCTGGATGTTGTTT"
                     /created_by="primer3"
                     /label="Uloop_detect_fow_wt"
     motif           complement(4054..4061)
                     /created_by="santi.bhattaraikline"
                     /label="Stem (RT Recognition)"
     primer_bind     complement(4085..4111)
                     /Mismatches=0
                     /%GC="55.6"
                     /Hairpin_Tm="57.4"
                     /Self_Dimer_Tm="14.9"
                     /Tm="68.4"
                     /annotation_group="1962289687766008e7df117-4f70-4fb3-a005-
                     defe567a0d26"
                     /Sequence="ATTCCGTATGCGCACCCTTAGCGAGAG"
                     /created_by="primer3"
                     /label="pSBK_106_4kld_fow"
     primer_bind     complement(4085..4111)
                     /Mismatches=0
                     /%GC="55.6"
                     /Hairpin_Tm="57.4"
                     /Self_Dimer_Tm="14.9"
                     /Tm="68.4"
                     /annotation_group="284591952286800a9cd0556-85ae-4e41-9dac-
                     d5ec59695a68"
                     /Sequence="ATTCCGTATGCGCACCCTTAGCGAGAG"
                     /created_by="primer3"
                     /label="106_4kld_fow"
     misc_feature    4092..4103
                     /created_by="User"
                     /Transferred_From="template"
                     /Transferred_Similarity="100.00%"
                     /label="'primer'"
     misc_signal     4105..>4118
                     /created_by="User"
                     /Transferred_From="template"
                     /Transferred_Similarity="100.00%"
                     /label="T7 to msr"
     source          complement(<4105..>4118)
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     4112..4139
                     /Mismatches=0
                     /%GC="50.0"
                     /Hairpin_Tm="51.3"
                     /Self_Dimer_Tm="None"
                     /Tm="65.5"
                     /annotation_group="196229170325000cb9946f6-0f94-4743-97b5-
                     f53dcee694fc"
                     /Sequence="CTTATCAGGAAAGCACATCCGGTGACAG"
                     /created_by="primer3"
                     /label="pSBK_106_4kld_rev"
     primer_bind     4112..4139
                     /Mismatches=0
                     /%GC="50.0"
                     /Hairpin_Tm="51.3"
                     /Self_Dimer_Tm="None"
                     /Tm="65.5"
                     /annotation_group="28459134964490048e7d26d-115c-44ba-be13-
                     3c869e4ed0dc"
                     /Sequence="CTTATCAGGAAAGCACATCCGGTGACAG"
                     /created_by="primer3"
                     /label="106_4kld_rev"
     misc_RNA        complement(<4119..4139)
                     /note="hammerhead ribozyme from the tobacco ringspot virus
                      satellite RNA (Khvorova et al., 2003)"
                     /label="sTRSV HHRz"
     promoter        complement(4142..4204)
                     /label="pBetI"
     promoter        complement(4148..4153)
                     /label="-10"
     operator        complement(4149..4170)
                     /label="BetO"
     promoter        complement(4171..4176)
                     /label="-35"
     primer_bind     complement(4188..48)
                     /modified_by="santi.bhattaraikline"
                     /Sequence="ATTAATAATCTGGCTTTTTATATTCTCTTAGAGCTGGGTTCGGACGC
                     GAGCGCGGGTGAGAGGGA"
                     /Mismatches=0
                     /annotation_group="674dfe0b-e430-4df4-aba9-9f2cbcf29c64"
                     /label="frag118_ins_rev_v2"
ORIGIN      
        1 cgcgtccgaa cccagctcta agagaatata aaaagccaga ttattaatcc ggctttttta
       61 ttatttcgct taacgatcgt tggctgtttt cagcaggacg cactgacctc cctatcagtg
      121 atagagattg acatccctat cagtgataga gatactgagc acagctgtca ccggatgtgc
      181 tttcctgata agattccgta tgcgcaccct tagcgagagg tttatcatta aggtcaacct
      241 ctggatgttg tttcggcatc ctgcattgaa tctgagttac aagaaaatgg agaggttgca
      301 gcaacctctc cacccgtttt ttctgacgta agggtgcgca tacggaatct tatcactcgg
      361 taccaaattc cagaaaagag gcctcccgaa aggggggcct tttttcgttt tggtccaatg
      421 gcggcgcgcc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
      481 gagtcaattc agggtggtga atatgtccag attagataaa agtaaagtga ttaacagcgc
      541 attagagctg cttaatgagg tcggaatcga aggtttaaca acccgtaaac tcgcccagaa
      601 gctaggtgta gagcagccta cattgtattg gcatgtaaaa aataagcggg ctttgctcga
      661 cgccttagcc attgagatgt tagataggca ccatactcac ttttgccctt tagaagggga
      721 aagctggcaa gattttttac gtaataacgc taaaagtttt agatgtgctt tactaagtca
      781 tcgcgatgga gcaaaagtac atttaggtac acggcctaca gaaaaacagt atgaaactct
      841 cgaaaatcaa ttagcctttt tatgccaaca aggtttttca ctagagaatg cattatatgc
      901 actcagcgct gtggggcatt ttactttagg ttgcgtattg gaagatcaag agcatcaagt
      961 cgctaaagaa gaaagggaaa cacctactac tgatagtatg ccgccattat tacgacaagc
     1021 tatcgaatta tttgatcacc aaggtgcaga gccagccttc ttattcggcc ttgaattgat
     1081 catatgcgga ttagaaaaac aacttaaatg tgaaagtggg tcctgataag gatcctaatt
     1141 ggtaacgaat cagaattgac ggctcgaggg agtagcatag ggtttgcaga atccctgctt
     1201 cgtccatttg acaggcacat tatgcatcga tgataagctg tcaaacatga gcagatcctc
     1261 tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat
     1321 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcaaatat
     1381 tttatctgag gtgcttcctc gctcactgct gaaaggagga actatatccg ggtaacgaat
     1441 tcaagcttga tatcattcag gacgagcctc agactccagc gtaactggac tgcaatcaac
     1501 tcactggctc accttcacgg gtgggccttt cttcggtaga aaatcaaagg atcttcttga
     1561 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1621 gtggtttgtt tgccggatca agagctacca actctttttc cgaggtaact ggcttcagca
     1681 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     1741 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     1801 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     1861 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1921 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     1981 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     2041 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     2101 atcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcag
     2161 aaaggcccac ccgaaggtga gccaggtgat tacatttggg ccctcatcag aggttttcac
     2221 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
     2281 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
     2341 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcattt
     2401 acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat
     2461 ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc
     2521 cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca
     2581 cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct
     2641 caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat
     2701 atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt
     2761 cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac
     2821 cgtctttcat tgccatacga aattccggat gagcattcat caggcgggca agaatgtgaa
     2881 taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat
     2941 ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt
     3001 ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccatag
     3061 ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt
     3121 gaaagttgga acctcttacg tgccgatcaa gtcaaaagcc tccggtcgga ggcttttgac
     3181 tttctgctat ggaggtcagg tatgatttaa atgtcaatcg gtcggcagat gctgggtgat
     3241 aaaatgacgg gtcaggctat ttgcacgggt tttatccagc ggtttaccgc tcagtgctgc
     3301 acgcagccac agaccatcaa tcagtgctgc cagaccataa cctgcctctt gtgcctgttc
     3361 acgaggcagt tcacgacgaa attcgctaac cagattgctc agcagacgac gactgctaac
     3421 ctgctgcaga cgatacagca tcggctgatg catgctgatt gcccaaaatg ccagccatgc
     3481 tttcattgct gcgctgctaa cctgggtttc atcaaaatta ccaccaacaa ttgcctgcag
     3541 acgctgttct gcgctaccct gcggcagtgc atgcagacga ttcagaactg catcacgcag
     3601 ctggctggta atatcacgca tggttgcttc cagtagaccg tttttatcgc ggaaataatg
     3661 gctaataata ccggtgctaa caccggcacg acgtgcaatc tgtgcaatgg ttgcatcatg
     3721 cataccaact tcattaattg cttccagggt tgcatcaatc agctgacgac gacgaatgct
     3781 ctgcataccc agtttcggca tattcacccc cgtgaattga ctctcttccg ggcgctatca
     3841 tgccataccg cgaaaggttt tgcaccattc gatggcgcgc cgccattgga ccaaaacgaa
     3901 aaaaggcccc cctttcggga ggcctctttt ctggaatttg gtaccgagtg ataagattcc
     3961 gtatgcgcac ccttacgtca gaaaaaacgg gtggagaggt cagtctgacc tctccatttt
     4021 cttgtaactc agattcaatg caggatgccg aaacaacatc cagaggttga ccttaatgat
     4081 aaacctctcg ctaagggtgc gcatacggaa tcttatcagg aaagcacatc cggtgacagc
     4141 tgctagcatt atattgaacg tccaatcaat tgtctattgg taacgaatcc ctctcacccg
     4201 cgct
//
