LOCUS       40924_37658        7077 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRR13, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7077)
  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 7077)
  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 7077)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7077)
  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..7077
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        95..113
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    114..138
                     /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)."
     RBS             153..175
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             194..211
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             227..937
                     /codon_start=1
                     /label=GFP (S65T)
                     /note="S65T variant of Aequorea victoria green fluorescent 
                     protein (Heim et al., 1995)"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             1352..1999
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe his5"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=S. pombe his5
                     /note="yeast auxotrophic marker; corresponds to S.
                     cerevisiae HIS3"
                     /translation="GRRAFVERNTNETKISVAIALDKAPLPEESNFIDELITSKHANQK
                     GEQVIQVDTGIGFLDHMYHALAKHAGWSLRLYSRGDLIIDDHHTAEDTAIALGIAFKQA
                     MGNFAGVKRFGHAYCPLDEALSRSVVDLSGRPYAVIDLGLKREKVGELSCEMIPHLLYS
                     FSVAAGITLHVTCLYGSNDHHRAESAFKSLAVAMRAATSLTGSSEVPSTKGVL"
     CDS             2165..2188
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     terminator      2230..2277
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2314..2769
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2887..3744)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(3745..3837)
                     /label=AmpR promoter
     rep_origin      3918..4506
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4692..4834)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4939..5127)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5665..5686)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5702..6781)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6782..6859)
                     /label=lacI promoter
ORIGIN
        1 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
       61 tagaggatcg agatcgatct cgatcccgcg aaattaatac gactcactat aggggaattg
      121 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
      181 catgggcagc agccatcacc atcatcacca cagccaggat cgatctagta aaggagaaga
      241 acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa
      301 attttctgtc agtggagagg gtgaaggtga tgcaacatac ggaaaactta cccttaaatt
      361 tatttgcact actggaaaac tacctgttcc atggccaaca cttgtcacta ctttcactta
      421 tggtgttcaa tgcttttcaa gatacccaga tcatatgaaa cggcatgact ttttcaagag
      481 tgccatgccc gaaggttatg tacaggaaag aactatattt ttcaaagatg acgggaacta
      541 caagacacgt gctgaagtca agtttgaagg tgataccctt gttaatagaa tcgagttaaa
      601 aggtattgat tttaaagaag atggaaacat tcttggacac aaattggaat acaactataa
      661 ctcacacaat gtatacatca tggcagacaa acaaaagaat ggaatcaaag ttaacttcaa
      721 aattagacac aacattgaag atggaagcgt tcaactagca gaccattatc aacaaaatac
      781 tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcca cacaatctgc
      841 cctttcgaaa gatcccaacg aaaagagaga ccacatggtc cttcttgagt ttgtaacagc
      901 tgctgggatt acacatggca tggatgaact atacaaaggt gctggtgcag gtgcaggagc
      961 tggagcaggt ttctgggaga aagcgtgttt ggccaaggga acccgtttgt tgagatgcga
     1021 tggaaccgag atcaatgtgg aagacgtgcg cgaaggtgac ctacttctgg gtcccgatgg
     1081 agagcctcgc cgtgcattca acatagtgaa tggcatcgac agactgtaca gaatcaagat
     1141 cggcggtgag aaagaggacc ttgtggtgac gccgaaccat attctggtgc tttatagaga
     1201 ggatggttcc aagaatgtgg agaagcaaac ggtggagatc actgctgccg agtttgccgc
     1261 gctttctacc gaggaaagaa gcctctatag tgcctttaca tctcctaggg ctgagaaggg
     1321 cgccgcaggt gcaggtgcag gtgcagctag cggtaggagg gcttttgtag aaagaaatac
     1381 gaacgaaacg aaaatcagcg ttgccatcgc tttggacaaa gctcccttac ctgaagagtc
     1441 gaattttatt gatgaactta taacttccaa gcatgcaaac caaaagggag aacaagtaat
     1501 ccaagtagac acgggaattg gattcttgga tcacatgtat catgcactgg ctaaacatgc
     1561 aggctggagc ttacgacttt actcaagagg tgatttaatc atcgatgatc atcacactgc
     1621 agaagatact gctattgcac ttggtattgc attcaagcag gctatgggta actttgccgg
     1681 cgttaaaaga tttggacatg cttattgtcc acttgacgaa gctctttcta gaagcgtagt
     1741 tgacttgtcg ggacggccct atgctgttat cgatttggga ttaaagcgtg aaaaggttgg
     1801 ggaattgtcc tgtgaaatga tccctcactt actatattcc ttttcggtag cagctggaat
     1861 tactttgcat gttacctgct tatatggtag taatgaccat catcgtgctg aaagcgcttt
     1921 taaatctctg gctgttgcca tgcgcgcggc tactagtctt actggaagtt ctgaagtccc
     1981 aagcacgaag ggagtgttgg gtaccgcggg tgctggtgct ggtgcttcgg ctcaaacgca
     2041 cagtttcaag attgagcaag ttagcctcga atccgagaag acagagtggg ctggtttccg
     2101 agtcgacaaa gatcagcttt acctgcgtca tgactacctt gtcctgcaca acagtggttt
     2161 cgaagattac aaggatgacg acgataagta agttaattaa cctaggctgc tgccaccgct
     2221 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
     2281 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
     2341 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     2401 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     2461 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     2521 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     2581 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     2641 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     2701 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     2761 ttacaatttc tggcggcacg atggcatgag attatcaaaa aggatcttca cctagatcct
     2821 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     2881 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     2941 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3001 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     3061 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3121 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3181 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3241 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3301 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3361 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3421 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     3481 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     3541 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3601 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3661 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3721 gacacggaaa tgttgaatac tcatactctt cctttttcaa tcatgattga agcatttatc
     3781 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3841 gtcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     3901 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     3961 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     4021 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     4081 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     4141 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     4201 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     4261 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     4321 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     4381 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     4441 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     4501 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     4561 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     4621 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     4681 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4741 atatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     4801 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4861 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4921 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4981 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     5041 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     5101 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     5161 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     5221 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     5281 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     5341 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     5401 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     5461 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     5521 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     5581 catgctagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     5641 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     5701 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5761 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     5821 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     5881 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     5941 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
     6001 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     6061 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     6121 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     6181 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     6241 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     6301 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     6361 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     6421 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     6481 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     6541 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     6601 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     6661 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     6721 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     6781 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     6841 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     6901 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     6961 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     7021 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcg
//
