LOCUS       40924_37663        7452 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRR14, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7452)
  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 7452)
  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 7452)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7452)
  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..7452
                     /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             1355..2374
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2540..2563
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     terminator      2605..2652
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2689..3144
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(3262..4119)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4120..4212)
                     /label=AmpR promoter
     rep_origin      4293..4881
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(5067..5209)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(5314..5502)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(6040..6061)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(6077..7156)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7157..7234)
                     /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 cggtaaaaag cctgaactca ccgcgacgtc
     1381 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     1441 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     1501 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     1561 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
     1621 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     1681 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     1741 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     1801 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     1861 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     1921 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     1981 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     2041 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     2101 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     2161 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     2221 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     2281 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     2341 ccgacgcccc agcactcgtc cgagggcaaa ggaaggtacc gcgggtgctg gtgctggtgc
     2401 ttcggctcaa acgcacagtt tcaagattga gcaagttagc ctcgaatccg agaagacaga
     2461 gtgggctggt ttccgagtcg acaaagatca gctttacctg cgtcatgact accttgtcct
     2521 gcacaacagt ggtttcgaag attacaagga tgacgacgat aagtaagtta attaacctag
     2581 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     2641 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     2701 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     2761 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     2821 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2881 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2941 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3001 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     3061 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     3121 acaaaatatt aacgtttaca atttctggcg gcacgatggc atgagattat caaaaaggat
     3181 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3241 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3301 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3361 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     3421 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3481 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3541 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3601 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3661 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3721 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3781 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3841 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     3901 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     3961 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     4021 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     4081 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatcatg
     4141 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     4201 aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4261 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4321 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4381 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4441 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4501 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4561 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4621 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4681 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4741 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4801 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     4861 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     4921 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4981 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     5041 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     5101 ggtatttcac accgcatata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     5161 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     5221 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     5281 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     5341 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     5401 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     5461 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     5521 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     5581 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     5641 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     5701 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     5761 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     5821 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     5881 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     5941 cgacaggagc acgatcatgc tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     6001 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     6061 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     6121 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     6181 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     6241 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     6301 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg
     6361 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     6421 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     6481 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     6541 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     6601 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     6661 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     6721 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     6781 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     6841 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     6901 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     6961 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     7021 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     7081 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     7141 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     7201 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     7261 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     7321 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     7381 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     7441 gtgatgtcgg cg
//
