LOCUS       40924_37628        6352 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pRR02, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6352)
  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 6352)
  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 6352)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6352)
  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..6352
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        183..211
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             918..935
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     CDS             1317..2120
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="GKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKPD
                     APELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTAF
                     QVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASDF
                     DDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIADR
                     YQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2295..2312
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     promoter        2655..2759
                     /label=AmpR promoter
     CDS             2760..3617
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3791..4379
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        4623..4700
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4701..5780
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    5796..5817
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5832..5862
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5870..5886
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     CDS             5906..6079
                     /codon_start=1
                     /label=lacZ-alpha
                     /note="LacZ-alpha fragment of beta-galactosidase"
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGE"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatccttctg ggagaaagcg tgtttggcca
      961 agggaacccg tttgttgaga tgcgatggaa ccgagatcaa tgtggaagac gtgcgcgaag
     1021 gtgacctact tctgggtccc gatggagagc ctcgccgtgc attcaacata gtgaatggca
     1081 tcgacagact gtacagaatc aagatcggcg gtgagaaaga ggaccttgtg gtgacgccga
     1141 accatattct ggtgctttat agagaggatg gttccaagaa tgtggagaag caaacggtgg
     1201 agatcactgc tgccgagttt gccgcgcttt ctaccgagga aagaagcctc tatagtgcct
     1261 ttacatctcc tagggctgag aagggcgccg caggtgcagg tgcaggtgca gctagcggta
     1321 aggaaaagac tcacgtttcg aggccgcgat taaattccaa catggatgct gatttatatg
     1381 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg
     1441 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     1501 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     1561 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccggcaaaa
     1621 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     1681 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     1741 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     1801 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataagc
     1861 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     1921 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     1981 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     2041 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     2101 tgatgctcga tgagtttttc ggtaccgcgg gtgctggtgc tggtgcttcg gctcaaacgc
     2161 acagtttcaa gattgagcaa gttagcctcg aatccgagaa gacagagtgg gctggtttcc
     2221 gagtcgacaa agatcagctt tacctgcgtc atgactacct tgtcctgcac aacagtggtt
     2281 tcgaaagagg atctcaccat caccatcacc atggatcccc ggaattcccg ggtcgactcg
     2341 agcggccgca tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat gacggtgaaa
     2401 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     2461 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     2521 cccagtcacg tagcgatagc ggagtgtata attcttgaag acgaaagggc ctcgtgatac
     2581 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     2641 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2701 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2761 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2821 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     2881 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     2941 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     3001 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     3061 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     3121 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     3181 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     3241 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     3301 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     3361 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     3421 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     3481 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3541 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3601 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     3661 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3721 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3781 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3841 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3901 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     3961 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4021 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4081 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4141 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4201 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4261 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4321 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4381 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4441 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4501 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4561 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcataaatt
     4621 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
     4681 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
     4741 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
     4801 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
     4861 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
     4921 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
     4981 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
     5041 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
     5101 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
     5161 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
     5221 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
     5281 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
     5341 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
     5401 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
     5461 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
     5521 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
     5581 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     5641 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     5701 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     5761 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     5821 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     5881 taacaatttc acacaggaaa cagctatgac catgattacg gattcactgg ccgtcgtttt
     5941 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     6001 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     6061 gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg gcaccagaag cggtgccgga
     6121 aagctggctg gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct caaactggca
     6181 gatgcacggt tacgatgcgc ccatctacac caacgtaacc tatcccatta cggtcaatcc
     6241 gccgtttgtt cccacggaga atccgacggg ttgttactcg ctcacattta atgttgatga
     6301 aagctggcta caggaaggcc agacgcgaat tatttttgat ggcgttggaa tt
//
