LOCUS       pCold MBP                5505 bp ds-DNA     circular SYN 22-七月-2016
DEFINITION  synthetic circular DNA
KEYWORDS    pCold MBP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5505)
  TITLE     Direct Submission
            //gt12365.cn/
FEATURES             Location/Qualifiers
     source          1..5505
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    15..81
                     /note="cspA promoter"
     protein_bind    84..100
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="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)."
     misc_feature    122..255
                     /note="cspA 5'UTR"
     misc_feature    256..270
                     /note="TEE"
     misc_feature    256..258
                     /note="M"
     CDS             271..288
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             289..300
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /note="Factor Xa site"
                     /translation="IEGR"
     CDS             304..1404
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /note="MBP"
                     /note="This version of the gene does not encode a signal
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     misc_feature    1405..1458
                     /note="multiple cloning site"
     misc_feature    1466..1610
                     /note="cspA 3'UTR"
     misc_feature    1494..1511
                     /note="transcription terminator"
     rep_origin      complement(1809..2264)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2364..2468
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             2469..3329
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      3500..4088
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(4260..5342)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(5343..5420)
                     /gene="lacI"
                     /note="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 catatgaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac
      361 ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag
      421 catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac
      481 attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa
      541 atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt
      601 tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac
      661 aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa
      721 ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg
      781 ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt
      841 aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg
      901 attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat
      961 aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc
     1021 aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc
     1081 gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag
     1141 ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg
     1201 ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg tgaaagatcc gcgtattgcc
     1261 gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct
     1321 ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat
     1381 gaagccctga aagacgcgca gactgagctc ggtaccctcg agggatccga attcaagctt
     1441 gtcgacctgc agtctagata ggtaatctct gcttaaaagc acagaatcta agatccctgc
     1501 catttggcgg ggattttttt atttgttttc aggaaataaa taatcgatcg cgtaataaaa
     1561 tctattatta tttttgtgaa gaataaattt gggtgcaatg agaatgcgca ggccctttcg
     1621 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     1681 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     1741 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     1801 gcaccataaa attgtaaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     1861 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     1921 gcccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     1981 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     2041 atcacccaaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     2101 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     2161 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     2221 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtactatg gttgctttga
     2281 cgtatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgtcag
     2341 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2401 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2461 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2521 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2581 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2641 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2701 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2761 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2821 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     2881 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     2941 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     3001 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     3061 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3121 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3181 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3241 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3301 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3361 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3421 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3481 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3541 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3601 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     3661 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3721 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3781 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3841 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3901 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3961 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4021 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4081 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4141 ctcacatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     4201 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     4261 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4321 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     4381 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     4441 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     4501 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     4561 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     4621 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     4681 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     4741 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     4801 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     4861 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     4921 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     4981 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     5041 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     5101 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     5161 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     5221 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     5281 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     5341 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     5401 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     5461 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgc
//