LOCUS       62056_7845        4930 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 4930)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4930
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        15..81
                     /label=cspA promoter
                     /note="promoter of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     protein_bind    84..100
                     /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)."
     5'UTR           122..255
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     CDS             256..270
                     /codon_start=1
                     /label=TEE
                     /note="translation enhancing element for E. coli (Qing et
                     al., 2004)"
                     /translation="MNHKV"
     CDS             280..303
                     /codon_start=1
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
                     /translation="WSHPQFEK"
     CDS             325..342
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             364..654
                     /codon_start=1
                     /label=SUMO
                     /note="cleavable ubiquitin-like protein tag"
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     CDS             676..702
                     /codon_start=1
                     /label=9xHis
                     /note="9xHis affinity tag"
                     /translation="HHHHHHHHH"
     CDS             706..729
                     /codon_start=1
                     /label=HRV 3C site
                     /note="recognition and cleavage site for human rhinovirus
                     3C and PreScission proteases"
                     /translation="LEVLFQGP"
     CDS             775..798
                     /codon_start=1
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
                     /translation="WSHPQFEK"
     CDS             850..873
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     3'UTR           890..1034
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     rep_origin      complement(1234..1689)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1789..1893
                     /label=AmpR promoter
     CDS             1894..2751
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2925..3513
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    complement(3651..3672)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3688..4767)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(4768..4845)
                     /label=lacI promoter
ORIGIN
        1 aaggaatggt gtggccgatt aatcataaat atgaaaaata attgttgcat cacccgccaa
       61 tgcgtggctt aatgcacatc aaattgtgag cggataacaa tttgatgtgc tagcgcatat
      121 ccagtgtagt aaggcaagtc ccttcaagag ttatcgttga tacccctcgt agtgcacatt
      181 cctttaacgc ttcaaaatct gtaaagcacg ccatatcgcc gaaaggcaca cttaattatt
      241 aagaggtaat acaccatgaa tcacaaagtg catatgaatt ggagccaccc gcagttcgaa
      301 aaaagcagcg gcagcagcgg cggtcatcac catcatcatc acggcggcag cggcggcagc
      361 gggtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct
      421 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa
      481 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg
      541 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcaggc ccctgaagat
      601 ttggacatgg aggataacga tattattgag gctcaccgcg aacagattgg aggcctggag
      661 ctcggtaccc tcgagcatca ccatcaccat caccatcacc atcacctgga agttctgttc
      721 caggggcccg aattctatgg caggaagaag cggagacagc gacgaagagg atcctggagc
      781 cacccgcagt tcgagaaagc ggccgcatat ggcaggaaga agcggagaca gcgacgaaga
      841 taagtcgacg attacaagga tgacgacgat aagtaatcta gataggtaat ctctgcttaa
      901 aagcacagaa tctaagatcc ctgccattgg gcggggattt ttttatttgt tttcaggaaa
      961 taaataatcg atcgcgtaat aaaatctatt attatttttg tgaagaataa atttgggtgc
     1021 aatgagaatg cgcaggccct ttcgtctcgc gccgtttcgg tgatgacggt gaaaacctct
     1081 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     1141 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     1201 catcagagca gattgtactg agagtgcacc ataaaattgt aaacgttaat attttgttaa
     1261 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     1321 aaatccctta taaatcaaaa gaatagcccg agatagggtt gagtgttgtt ccagtttgga
     1381 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     1441 agggcgatgg cccactacgt gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc
     1501 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     1561 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     1621 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     1681 agggcgcgta ctatggttgc tttgacgtat gcggtgtgaa ataccgcaca gatgcgtaag
     1741 gagaaaatac cgcatcaggc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     1801 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     1861 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     1921 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     1981 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2041 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2101 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2161 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2221 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2281 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2341 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     2401 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     2461 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     2521 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     2581 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     2641 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     2701 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     2761 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     2821 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     2881 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2941 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3001 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3061 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3121 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3181 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3241 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3301 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3361 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     3421 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     3481 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     3541 ttcctggcct tttgctggcc ttttgctcac atagtcatgc cccgcgccca ccggaaggag
     3601 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     3661 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     3721 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     3781 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     3841 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     3901 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     3961 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     4021 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     4081 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     4141 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     4201 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     4261 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     4321 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     4381 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     4441 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     4501 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     4561 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     4621 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     4681 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     4741 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     4801 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     4861 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     4921 ccgccgccgc
//
