LOCUS       62056_7850        5643 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 5643)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5643
                     /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             271..288
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             289..300
                     /codon_start=1
                     /label=Factor Xa site
                     /note="Factor Xa recognition and cleavage site"
                     /translation="IEGR"
     CDS             340..633
                     /codon_start=1
                     /label=SUMO
                     /note="cleavable ubiquitin-like protein tag"
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             640..1587
                     /codon_start=1
                     /label=iRFP720
                     /note="near-infrared fluorescent protein with an emission
                     peak at 720 nm, engineered from a bacterial phytochrome 
                     (Shcherbakova and Verkhusha, 2013)"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     3'UTR           1604..1748
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene
                     (Mitta et al., 1997)"
     rep_origin      complement(1947..2402)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2502..2606
                     /label=AmpR promoter
     CDS             2607..3464
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3638..4226
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    complement(4364..4385)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(4401..5480)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(5481..5558)
                     /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 catcatcatc atcatcatat cgaaggtagg
      301 catatgggcg gtggcggtag cggcggtggc ggtagcggta tgtcggactc agaagtcaat
      361 caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat caatttaaag
      421 gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc tttaagaagg
      481 ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag attcttgtac
      541 gacggtatta gaattcaagc tgatcagacc cctgaagatt tggacatgga ggataacgat
      601 attattgagg ctcacagaga acagattggt ggtactagta tggcggaagg atccgtcgcc
      661 aggcagcctg acctcttgac ctgcgacgat gagccgatcc atatccccgg tgccatccaa
      721 ccgcatggac tgctgctcgc cctcgccgcc gacatgacga tcgttgccgg cagcgacaac
      781 cttcccgaac tcaccggact ggcgatcggc gccctgatcg gccgctctgc ggccgatgtc
      841 ttcgactcgg agacgcacaa ccgtctgacg atcgccttgg ccgagcccgg ggcggccgtc
      901 ggagcaccga tcactgtcgg cttcacgatg cgaaaggacg caggcttcat cggctcctgg
      961 catcgccatg atcagctcat cttcctcgag ctcgagcctc cccagcggga cgtcgccgag
     1021 ccgcaggcgt tcttccgccg caccaacagc gccatccgcc gcctgcaggc cgccgaaacc
     1081 ttggaaagcg cctgcgccgc cgcggcgcaa gaggtgcgga agattaccgg cttcgatcgg
     1141 gtgatgatct atcgcttcgc ctccgacttc agcgggtccg tgatcgcaga ggatcggtgc
     1201 gccgaggtcg agtcaaaact aggcctgcac tatcctgcct cattcatccc ggcgcaggcc
     1261 cgtcggctct ataccatcaa cccggtacgg atcattcccg atatcaatta tcggccggtg
     1321 ccggtcaccc cagacctcaa tccggtcacc gggcggccga ttgatcttag cttcgccatc
     1381 ctgcgcagcg tctcgcccaa ccatctggag ttcatgcgca acataggcat gcacggcacg
     1441 atgtcgatct cgattttgcg cggcgagcga ctgtggggat tgatcgtttg ccatcaccga
     1501 acgccgtact acgtcgatct cgatggccgc caagcctgcg agctagtcgc ccaggttctg
     1561 gcctggcaga tcggcgtgat ggaagagtag tctagatagg taatctctgc ttaaaagcac
     1621 agaatctaag atccctgcca tttggcgggg atttttttat ttgttttcag gaaataaata
     1681 atcgatcgcg taataaaatc tattattatt tttgtgaaga ataaatttgg gtgcaatgag
     1741 aatgcgcagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     1801 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     1861 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     1921 gcagattgta ctgagagtgc accataaaat tgtaaacgtt aatattttgt taaaattcgc
     1981 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2041 ttataaatca aaagaatagc ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2101 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2161 tggcccacta cgtgaaccat cacccaaatc aagttttttg gggtcgaggt gccgtaaagc
     2221 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2281 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2341 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2401 gtactatggt tgctttgacg tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     2461 taccgcatca ggcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2521 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2581 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     2641 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     2701 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     2761 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     2821 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     2881 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     2941 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3001 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3061 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3121 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3181 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3241 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     3301 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     3361 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     3421 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     3481 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     3541 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     3601 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3661 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3721 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3781 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3841 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3901 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3961 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4021 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4081 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4141 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4201 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4261 ccttttgctg gccttttgct cacatagtca tgccccgcgc ccaccggaag gagctgactg
     4321 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     4381 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4441 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     4501 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     4561 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     4621 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     4681 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     4741 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     4801 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     4861 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     4921 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     4981 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     5041 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     5101 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     5161 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     5221 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     5281 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     5341 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     5401 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     5461 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     5521 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     5581 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     5641 cgc
//
