LOCUS       62056_10095        6253 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6253)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      12..467
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(563..1375)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      1497..2085
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        2133..2224
                     /label=AmpR promoter
     CDS             2225..3013
                     /codon_start=1
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     misc_feature    complement(3155..3297)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(3402..3590)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(4365..4386)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(4402..5481)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(5482..5559)
                     /label=lacI promoter
     promoter        5868..5886
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5887..5911
                     /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             5926..5948
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5967..5984
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             5994..6011
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     CDS             6015..6047
                     /codon_start=1
                     /label=T7 tag (gene 10 leader)
                     /note="leader peptide from bacteriophage T7 gene 10"
                     /translation="MASMTGGQQMG"
     CDS             6097..6114
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      6181..6228
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
       61 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
      121 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
      181 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
      241 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
      301 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
      361 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      421 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt
      481 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      541 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
      601 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
      661 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
      721 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
      781 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
      841 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
      901 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
      961 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     1021 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     1081 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     1141 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     1201 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     1261 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     1321 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     1381 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     1441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     1501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     1561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     1621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     1681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     1741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     1801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     1861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     1921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     1981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     2041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     2101 gcctttttac ggttcctggc cttttgctgg cctttgttta tttttctaaa tacattcaaa
     2161 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2221 gagtatgagg gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt
     2281 catcgagcgc catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga
     2341 tggcggcctg aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga
     2401 tgaaacaacg cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga
     2461 gagcgagatt ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg
     2521 gcgttatcca gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc
     2581 aggtatcttc gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag
     2641 agaacatagc gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga
     2701 acaggatcta tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg
     2761 ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac
     2821 cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggccca
     2881 gtatcagccc gtcatacttg aagctagaca ggcttatctt ggacaagaag aagatcgctt
     2941 ggcctcgcgc gcagatcagt tggaagaatt tgtccactac gtgaaaggcg agatcaccaa
     3001 ggtagtcggc aaataatttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3061 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3121 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3181 atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct gctctgatgc
     3241 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3301 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3361 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3421 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3481 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     3541 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     3601 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     3661 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     3721 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     3781 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     3841 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     3901 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     3961 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4021 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     4081 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4141 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     4201 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     4261 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     4321 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4381 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4441 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4501 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4561 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4621 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4681 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4741 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4801 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4861 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     4921 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     4981 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5041 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5101 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5161 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5221 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5281 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5341 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5401 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5461 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5521 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5581 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     5641 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     5701 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     5761 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     5821 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     5881 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     5941 gaaggagata taccatgggc agcagccatc atcatcatca tcacagcagc ggcctggtgc
     6001 cgcgcggcag ccatatggct agcatgactg gtggacagca aatgggtcgc ggatccgaat
     6061 tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
     6121 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     6181 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     6241 actatatccg gat
//
