LOCUS       40924_37643        6430 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRR05, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6430)
  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 6430)
  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 6430)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6430)
  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..6430
                     /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..1919
                     /codon_start=1
                     /label=LexA
                     /note="E. coli DNA-binding protein that represses genes
                     involved in the SOS response to DNA damage"
                     /translation="KALTARQQEVFDLIRDHISQTGMPPTRAEIAQRLGFRSPNAAEEH
                     LKALARKGVIEIVSGASRGIRLLQEEEEGLPLVGRVAAGEPLLAQQHIEGHYQVDPSLF
                     KPNADFLLRVSGMSMKDIGIMDGDLLAVHKTQDVRNGQVVVARIDDEVTVKRLKKQGNK
                     VELLPENSEFKPIVVDLRQQSFTIEGLAVGVIRNGDWL"
     CDS             1965..2198
                     /codon_start=1
                     /label=VP16 AD
                     /note="transcriptional activation domain of herpes simplex
                     virus protein VP16 (Triezenberg et al., 1988; Cousens et 
                     al., 1989)"
                     /translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
                     TPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     CDS             2373..2390
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     promoter        2733..2837
                     /label=AmpR promoter
     CDS             2838..3695
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3869..4457
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        4701..4778
                     /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             4779..5858
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    5874..5895
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5910..5940
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5948..5964
                     /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             5984..6157
                     /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 gctagcaaag
     1321 cgttaacggc caggcaacaa gaggtgtttg atctcatccg tgatcacatc agccagacag
     1381 gtatgccgcc gacgcgtgcg gaaatcgcgc agcgtttggg gttccgttcc ccaaacgcgg
     1441 ctgaagaaca tctgaaggcg ctggcacgca aaggcgttat tgaaattgtt tccggcgcat
     1501 cacgcgggat tcgtctgttg caggaagagg aagaagggtt gccgctggta ggtcgtgtgg
     1561 ctgccggtga accacttctg gcgcaacagc atattgaagg tcattatcag gtcgatcctt
     1621 ccttattcaa gccgaatgct gatttcctgc tgcgcgtcag cgggatgtcg atgaaagata
     1681 tcggcattat ggatggtgac ttgctggcag tgcataaaac tcaggatgta cgtaacggtc
     1741 aggtcgttgt cgcacgtatt gatgacgaag ttaccgttaa gcgcctgaaa aaacagggca
     1801 ataaagtcga actgttgcca gaaaatagcg agtttaaacc aattgtcgta gatcttcgtc
     1861 agcagagctt caccattgaa gggctggcgg ttggggttat tcgcaacggc gactggctgg
     1921 aattcccggg gatccgtcga cctgcaggaa ttcccgggga tctggccccc ccgaccgatg
     1981 tcagcctggg ggacgagctc cacttagacg gcgaggacgt ggcgatggcg catgccgacg
     2041 cgctagacga tttcgatctg gacatgttgg gggacgggga ttccccgggt ccgggattta
     2101 ccccccacga ctccgccccc tacggcgctc tggatatggc cgacttcgag tttgagcaga
     2161 tgtttaccga tgcccttgga attgacgagt acggtggggg taccgcgggt gctggtgctg
     2221 gtgcttcggc tcaaacgcac agtttcaaga ttgagcaagt tagcctcgaa tccgagaaga
     2281 cagagtgggc tggtttccga gtcgacaaag atcagcttta cctgcgtcat gactaccttg
     2341 tcctgcacaa cagtggtttc gaaagaggat ctcaccatca ccatcaccat ggatccccgg
     2401 aattcccggg tcgactcgag cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt
     2461 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     2521 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     2581 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac
     2641 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     2701 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2761 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2821 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     2881 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2941 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3001 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     3061 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     3121 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3181 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3241 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3301 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3361 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3421 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3481 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3541 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3601 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3661 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3721 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3781 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3841 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3901 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3961 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     4021 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     4081 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     4141 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4201 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4261 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4321 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4381 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4441 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4501 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4561 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4621 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4681 tttcacaccg cataaattcc gacaccatcg aatggtgcaa aacctttcgc ggtatggcat
     4741 gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg
     4801 atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
     4861 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca
     4921 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca
     4981 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
     5041 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta
     5101 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
     5161 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc
     5221 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc
     5281 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
     5341 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca
     5401 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
     5461 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc
     5521 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata
     5581 tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca
     5641 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct
     5701 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
     5761 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     5821 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
     5881 agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg
     5941 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga
     6001 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     6061 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     6121 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc
     6181 accagaagcg gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt
     6241 cgtcccctca aactggcaga tgcacggtta cgatgcgccc atctacacca acgtaaccta
     6301 tcccattacg gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct
     6361 cacatttaat gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg
     6421 cgttggaatt
//
