LOCUS       62056_20800        6901 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 6901)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6901
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    complement(1..140)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(245..433)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(1208..1229)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(1245..2324)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(2325..2402)
                     /label=lacI promoter
     promoter        2711..2729
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2730..2754
                     /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             2769..2791
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2865..3581
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             4362..4379
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             4428..4451
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     CDS             4476..4493
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      4560..4607
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4644..5099
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(5194..6006)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      6128..6716
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
       61 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
      121 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
      181 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
      241 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
      301 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
      361 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
      421 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
      481 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
      541 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
      601 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
      661 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
      721 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
      781 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
      841 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
      901 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
      961 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     1021 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     1081 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     1141 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     1201 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     1261 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1321 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     1381 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     1441 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     1501 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     1561 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     1621 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     1681 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     1741 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     1801 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     1861 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     1921 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     1981 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     2041 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     2101 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     2161 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     2221 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     2281 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     2341 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     2401 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     2461 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     2521 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     2581 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     2641 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     2701 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     2761 gaaataattt tgtttaactt taagaaggag atatacatat gaaatacctg ctgccgaccg
     2821 ctgctgctgg tctgctgctc ctcgctgccc agccggccat ggccatggtg agcaagggcg
     2881 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     2941 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     3001 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     3061 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     3121 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     3181 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     3241 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     3301 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     3361 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     3421 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     3481 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     3541 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gggaggaggg ggaagcgagg
     3601 tgcagctggt gcagtctggg gctgaggtga agaagcctgg ggcctcagtg aaggtctcct
     3661 gcaaggcttc tggttacacc tttaccagct atagtatcag ctgggtgcga caggcccctg
     3721 gacaagggct tgagtggatg ggatggatca gcgcttacaa tggtaacaca agctatgcac
     3781 agaagctcca gggcagagtc acgattaccg cggacaaatc cacgagcaca gcctacatgg
     3841 agctgagcag cctgagatct gaggacacgg ccgtgtatta ctgtgcgaag gccgggcacc
     3901 gttcagggag atataacaac tggttcgacc cctggggcca gggcaccctg gtcaccgtct
     3961 cctcactcga gggtggaggc ggttcaggcg gaggtggctc tggcggtggc gctagccagt
     4021 ctgagctgac tcagcctccc tcggtgtcag tggccccagg acagacggcc aggatcacct
     4081 gtggggaaaa caacattgga agtaaaaatg tgcactggta ccagcagaag ccaggccagg
     4141 cccctgtgct gatcatctat aggggtagca accggccctc tgggatccct gagcgattct
     4201 ctggctccaa ctcggggaac acggccaccc tgaccatcag cagagcccaa gccggggatg
     4261 aggctgacta ttactgccag gtgtttgata ggaggagtga ccatccggtg gtgttcggcg
     4321 gagggaccaa gctgaccgtc ctaggtgcgg ccgcatccgc acatcatcat caccatcaca
     4381 agctggacta caaagaccat gacggtgatt ataaagatca tgacatcgat tacaaggatg
     4441 acgatgacaa gtaataaaag ctttaataag tcgagcacca ccaccaccac cactgagatc
     4501 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     4561 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     4621 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4681 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4741 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4801 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4861 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4921 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4981 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     5041 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     5101 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5161 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     5221 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     5281 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     5341 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     5401 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     5461 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     5521 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     5581 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     5641 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     5701 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     5761 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     5821 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     5881 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     5941 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     6001 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     6061 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6121 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6181 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6241 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     6301 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6361 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6421 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6481 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6541 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6601 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6661 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6721 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6781 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     6841 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     6901 g
//
