LOCUS       40924_37648        6435 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRR11, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6435)
  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 6435)
  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 6435)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6435)
  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..6435
                     /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             1523..1546
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     terminator      1588..1635
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1672..2127
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2245..3102)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(3103..3195)
                     /label=AmpR promoter
     rep_origin      3276..3864
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4050..4192)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(4297..4485)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5023..5044)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5060..6139)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6140..6217)
                     /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 cgcagcaggt accgcgggtg ctggtgctgg
     1381 tgcttcggct caaacgcaca gtttcaagat tgagcaagtt agcctcgaat ccgagaagac
     1441 agagtgggct ggtttccgag tcgacaaaga tcagctttac ctgcgtcatg actaccttgt
     1501 cctgcacaac agtggtttcg aagattacaa ggatgacgac gataagtaag ttaattaacc
     1561 taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1621 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     1681 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1741 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1801 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1861 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1921 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1981 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     2041 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     2101 ttaacaaaat attaacgttt acaatttctg gcggcacgat ggcatgagat tatcaaaaag
     2161 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     2221 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     2281 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     2341 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     2401 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     2461 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     2521 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     2581 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     2641 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     2701 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     2761 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     2821 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     2881 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     2941 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     3001 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     3061 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaatc
     3121 atgattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3181 agaaaaataa acaaataggt catgaccaaa atcccttaac gtgagttttc gttccactga
     3241 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     3301 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     3361 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     3421 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     3481 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     3541 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3601 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3661 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3721 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3781 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3841 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3901 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3961 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     4021 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     4081 tgcggtattt cacaccgcat atatggtgca ctctcagtac aatctgctct gatgccgcat
     4141 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     4201 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4261 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4321 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     4381 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     4441 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     4501 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     4561 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     4621 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     4681 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     4741 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     4801 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     4861 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     4921 caacgacagg agcacgatca tgctagtcat gccccgcgcc caccggaagg agctgactgg
     4981 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     5041 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5101 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     5161 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5221 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     5281 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     5341 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     5401 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     5461 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5521 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     5581 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     5641 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     5701 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     5761 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     5821 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     5881 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     5941 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     6001 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     6061 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     6121 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     6181 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     6241 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     6301 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     6361 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     6421 tcggtgatgt cggcg
//
