LOCUS       40924_37633        6196 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRR03, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6196)
  AUTHORS   Ramsden R, Arms L, Davis TN, Muller EG.
  TITLE     An intein with genetically selectable markers provides a new 
            approach to internally label proteins with GFP
  JOURNAL   BMC Biotechnol. 11 (1), 71 (2011)
  PUBMED    21708017
REFERENCE   2  (bases 1 to 6196)
  AUTHORS   Ramsden R, Arms L, Davis TN, Muller EGD.
  TITLE     Direct Submission
  JOURNAL   Submitted (30-JUN-2011) Biochemistry, University of Washington, 1705
            NE Pacific St, HSB J461, Seattle, WA 98195, USA
REFERENCE   3  (bases 1 to 6196)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6196)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC 
            Biotechnol."; date: "2011"; volume: "11"; issue: "1"; pages: "71"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (30-JUN-2011) Biochemistry, University of Washington, 1705 NE 
            Pacific St, HSB J461, Seattle, WA 98195, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6196
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        183..211
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    219..235
                     /label=lac operator
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             918..935
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     CDS             1317..1964
                     /codon_start=1
                     /label=S. pombe his5
                     /note="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /translation="GRRAFVERNTNETKISVAIALDKAPLPEESNFIDELITSKHANQK
                     GEQVIQVDTGIGFLDHMYHALAKHAGWSLRLYSRGDLIIDDHHTAEDTAIALGIAFKQA
                     MGNFAGVKRFGHAYCPLDEALSRSVVDLSGRPYAVIDLGLKREKVGELSCEMIPHLLYS
                     FSVAAGITLHVTCLYGSNDHHRAESAFKSLAVAMRAATSLTGSSEVPSTKGVL"
     CDS             2139..2156
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     promoter        2499..2603
                     /label=AmpR promoter
     CDS             2604..3461
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3635..4223
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        4467..4544
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4545..5624
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    5640..5661
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5676..5706
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5714..5730
                     /label=lac operator
                     /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)."
     CDS             5750..5923
                     /codon_start=1
                     /label=lacZ-alpha
                     /note="LacZ-alpha fragment of beta-galactosidase"
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatccttctg ggagaaagcg tgtttggcca
      961 agggaacccg tttgttgaga tgcgatggaa ccgagatcaa tgtggaagac gtgcgcgaag
     1021 gtgacctact tctgggtccc gatggagagc ctcgccgtgc attcaacata gtgaatggca
     1081 tcgacagact gtacagaatc aagatcggcg gtgagaaaga ggaccttgtg gtgacgccga
     1141 accatattct ggtgctttat agagaggatg gttccaagaa tgtggagaag caaacggtgg
     1201 agatcactgc tgccgagttt gccgcgcttt ctaccgagga aagaagcctc tatagtgcct
     1261 ttacatctcc tagggctgag aagggcgccg caggtgcagg tgcaggtgca gctagcggta
     1321 ggagggcttt tgtagaaaga aatacgaacg aaacgaaaat cagcgttgcc atcgctttgg
     1381 acaaagctcc cttacctgaa gagtcgaatt ttattgatga acttataact tccaagcatg
     1441 caaaccaaaa gggagaacaa gtaatccaag tagacacggg aattggattc ttggatcaca
     1501 tgtatcatgc actggctaaa catgcaggct ggagcttacg actttactca agaggtgatt
     1561 taatcatcga tgatcatcac actgcagaag atactgctat tgcacttggt attgcattca
     1621 agcaggctat gggtaacttt gccggcgtta aaagatttgg acatgcttat tgtccacttg
     1681 acgaagctct ttctagaagc gtagttgact tgtcgggacg gccctatgct gttatcgatt
     1741 tgggattaaa gcgtgaaaag gttggggaat tgtcctgtga aatgatccct cacttactat
     1801 attccttttc ggtagcagct ggaattactt tgcatgttac ctgcttatat ggtagtaatg
     1861 accatcatcg tgctgaaagc gcttttaaat ctctggctgt tgccatgcgc gcggctacta
     1921 gtcttactgg aagttctgaa gtcccaagca cgaagggagt gttgggtacc gcgggtgctg
     1981 gtgctggtgc ttcggctcaa acgcacagtt tcaagattga gcaagttagc ctcgaatccg
     2041 agaagacaga gtgggctggt ttccgagtcg acaaagatca gctttacctg cgtcatgact
     2101 accttgtcct gcacaacagt ggtttcgaaa gaggatctca ccatcaccat caccatggat
     2161 ccccggaatt cccgggtcga ctcgagcggc cgcatcgtga ctgactgacg atctgcctcg
     2221 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     2281 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     2341 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tataattctt
     2401 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2461 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2521 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2581 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2641 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2701 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2761 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2821 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     2881 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2941 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3001 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3061 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3121 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     3181 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3241 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3301 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3361 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3421 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3481 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3541 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3601 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3661 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3721 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3781 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3841 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3901 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3961 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4021 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4081 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4141 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4201 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4261 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4321 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4381 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4441 gcggtatttc acaccgcata aattccgaca ccatcgaatg gtgcaaaacc tttcgcggta
     4501 tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt
     4561 tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc
     4621 aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga
     4681 attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg
     4741 ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc
     4801 gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag
     4861 cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact
     4921 atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt
     4981 tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg
     5041 gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag
     5101 cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca
     5161 ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt
     5221 ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca
     5281 acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg
     5341 cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt
     5401 taaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc
     5461 aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa
     5521 gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     5581 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     5641 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     5701 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     5761 tacggattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     5821 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     5881 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgct ttgcctggtt
     5941 tccggcacca gaagcggtgc cggaaagctg gctggagtgc gatcttcctg aggccgatac
     6001 tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat gcgcccatct acaccaacgt
     6061 aacctatccc attacggtca atccgccgtt tgttcccacg gagaatccga cgggttgtta
     6121 ctcgctcaca tttaatgttg atgaaagctg gctacaggaa ggccagacgc gaattatttt
     6181 tgatggcgtt ggaatt
//
