LOCUS       40924_37653        7233 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRR12, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7233)
  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 7233)
  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 7233)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7233)
  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..7233
                     /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..2152
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="GKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKPD
                     APELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTAF
                     QVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASDF
                     DDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIADR
                     YQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEF"
     CDS             2321..2344
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     terminator      2386..2433
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2470..2925
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(3043..3900)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(3901..3993)
                     /label=AmpR promoter
     rep_origin      4074..4662
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(4848..4990)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(5095..5283)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(5821..5842)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5858..6937)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(6938..7015)
                     /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 cggtaaggaa aagactcacg tttcgaggcc
     1381 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     1441 cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt
     1501 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     1561 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     1621 tgcatggtta ctcaccactg cgatccccgg caaaacagca ttccaggtat tagaagaata
     1681 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     1741 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     1801 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     1861 gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac cggattcagt
     1921 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     1981 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     2041 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     2101 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttgggtac
     2161 cgcgggtgct ggtgctggtg cttcggctca aacgcacagt ttcaagattg agcaagttag
     2221 cctcgaatcc gagaagacag agtgggctgg tttccgagtc gacaaagatc agctttacct
     2281 gcgtcatgac taccttgtcc tgcacaacag tggtttcgaa gattacaagg atgacgacga
     2341 taagtaagtt aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt
     2401 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     2461 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     2521 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     2581 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2641 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2701 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2761 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2821 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2881 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttctggc ggcacgatgg
     2941 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     3001 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     3061 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     3121 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     3181 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3241 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3301 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     3361 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     3421 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     3481 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3541 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3601 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3661 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     3721 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     3781 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     3841 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     3901 actcttcctt tttcaatcat gattgaagca tttatcaggg ttattgtctc atgagcggat
     3961 acatatttga atgtatttag aaaaataaac aaataggtca tgaccaaaat cccttaacgt
     4021 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4081 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4141 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4201 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     4261 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4321 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4381 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4441 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4501 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4561 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4621 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4681 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4741 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4801 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4861 tttctcctta cgcatctgtg cggtatttca caccgcatat atggtgcact ctcagtacaa
     4921 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4981 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5041 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5101 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     5161 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     5221 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     5281 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     5341 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     5401 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     5461 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     5521 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     5581 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     5641 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     5701 ccagcctagc cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca
     5761 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     5821 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5881 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5941 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     6001 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     6061 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     6121 atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     6181 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     6241 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     6301 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     6361 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     6421 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     6481 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     6541 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     6601 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     6661 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     6721 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     6781 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     6841 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     6901 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     6961 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     7021 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     7081 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     7141 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     7201 cgagcccgat cttccccatc ggtgatgtcg gcg
//
