LOCUS       40924_37668        7311 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRR15, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7311)
  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 7311)
  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 7311)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7311)
  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..7311
                     /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..1954
                     /codon_start=1
                     /gene="lexA"
                     /product="E. coli DNA-binding protein that represses genes
                     involved in the SOS response to DNA damage"
                     /label=LexA
                     /note="The N-terminal DNA-binding domain recognizes the
                     16-bp palindromic sequence 5'-CTGTATATATATACAG-3'."
                     /translation="KALTARQQEVFDLIRDHISQTGMPPTRAEIAQRLGFRSPNAAEEH
                     LKALARKGVIEIVSGASRGIRLLQEEEEGLPLVGRVAAGEPLLAQQHIEGHYQVDPSLF
                     KPNADFLLRVSGMSMKDIGIMDGDLLAVHKTQDVRNGQVVVARIDDEVTVKRLKKQGNK
                     VELLPENSEFKPIVVDLRQQSFTIEGLAVGVIRNGDWL"
     CDS             2000..2233
                     /codon_start=1
                     /gene="UL48"
                     /product="transcriptional activation domain of herpes
                     simplex virus protein VP16 (Triezenberg et al., 1988; 
                     Cousens et al., 1989)"
                     /label=VP16 AD
                     /translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
                     TPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     CDS             2399..2422
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     terminator      2464..2511
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2548..3003
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(3121..3978)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(3979..4071)
                     /label=AmpR promoter
     rep_origin      4152..4740
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4926..5068)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(5173..5361)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5899..5920)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5936..7015)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7016..7093)
                     /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 caaagcgtta acggccaggc aacaagaggt
     1381 gtttgatctc atccgtgatc acatcagcca gacaggtatg ccgccgacgc gtgcggaaat
     1441 cgcgcagcgt ttggggttcc gttccccaaa cgcggctgaa gaacatctga aggcgctggc
     1501 acgcaaaggc gttattgaaa ttgtttccgg cgcatcacgc gggattcgtc tgttgcagga
     1561 agaggaagaa gggttgccgc tggtaggtcg tgtggctgcc ggtgaaccac ttctggcgca
     1621 acagcatatt gaaggtcatt atcaggtcga tccttcctta ttcaagccga atgctgattt
     1681 cctgctgcgc gtcagcggga tgtcgatgaa agatatcggc attatggatg gtgacttgct
     1741 ggcagtgcat aaaactcagg atgtacgtaa cggtcaggtc gttgtcgcac gtattgatga
     1801 cgaagttacc gttaagcgcc tgaaaaaaca gggcaataaa gtcgaactgt tgccagaaaa
     1861 tagcgagttt aaaccaattg tcgtagatct tcgtcagcag agcttcacca ttgaagggct
     1921 ggcggttggg gttattcgca acggcgactg gctggaattc ccggggatcc gtcgacctgc
     1981 aggaattccc ggggatctgg cccccccgac cgatgtcagc ctgggggacg agctccactt
     2041 agacggcgag gacgtggcga tggcgcatgc cgacgcgcta gacgatttcg atctggacat
     2101 gttgggggac ggggattccc cgggtccggg atttaccccc cacgactccg ccccctacgg
     2161 cgctctggat atggccgact tcgagtttga gcagatgttt accgatgccc ttggaattga
     2221 cgagtacggt gggggtaccg cgggtgctgg tgctggtgct tcggctcaaa cgcacagttt
     2281 caagattgag caagttagcc tcgaatccga gaagacagag tgggctggtt tccgagtcga
     2341 caaagatcag ctttacctgc gtcatgacta ccttgtcctg cacaacagtg gtttcgaaga
     2401 ttacaaggat gacgacgata agtaagttaa ttaacctagg ctgctgccac cgctgagcaa
     2461 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     2521 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     2581 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2641 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2701 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2761 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     2821 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     2881 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2941 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     3001 tttctggcgg cacgatggca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     3061 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3121 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3181 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     3241 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     3301 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     3361 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     3421 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     3481 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     3541 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     3601 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3661 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3721 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     3781 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     3841 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     3901 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     3961 gaaatgttga atactcatac tcttcctttt tcaatcatga ttgaagcatt tatcagggtt
     4021 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggtcatg
     4081 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4141 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4201 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4261 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     4321 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4381 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4441 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4501 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4561 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4621 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4681 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4741 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4801 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4861 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     4921 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatat
     4981 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     5041 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     5101 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     5161 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     5221 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     5281 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     5341 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     5401 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     5461 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     5521 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     5581 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     5641 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     5701 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     5761 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgct
     5821 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     5881 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     5941 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     6001 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     6061 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     6121 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     6181 tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact
     6241 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     6301 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     6361 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     6421 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     6481 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     6541 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     6601 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     6661 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     6721 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     6781 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     6841 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     6901 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     6961 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     7021 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     7081 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     7141 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     7201 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     7261 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc g
//
