LOCUS       40924_355        7180 bp DNA     circular SYN 13-MAY-2021
DEFINITION  E. coli expression, protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7180)
  AUTHORS   Teng F, Cui T, Feng G, Guo L, Xu K, Gao Q, Li T, Li J, Zhou Q, Li W
  TITLE     Repurposing CRISPR-Cas12b for mammalian genome engineering.
  JOURNAL   Cell Discov. 2018 Nov 27;4:63. doi: 10.1038/s41421-018-0069-3. 
            eCollection 2018.
  PUBMED    30510770
REFERENCE   2  (bases 1 to 7180)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7180)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell
            Discov."; date: "2018-11-27"; pages: "
            10.1038/s41421-018-0069-3. eCollection 2018"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    12..36
                     /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             51..73
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..103
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             3503..3529
                     /codon_start=1
                     /label=9xHis
                     /note="9xHis affinity tag"
                     /translation="HHHHHHHHH"
     promoter        3601..3619
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      3641..3688
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3912..4568)
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(4569..4671)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5063..5080)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5197..5742)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     protein_bind    complement(5933..5954)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(5970..7049)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(7050..7127)
                     /label=lacI promoter
ORIGIN
        1 ctcactatag gggaattgtg agcggataac aattcccctg tagaaataat tttgtttaac
       61 tttaataagg agatatcata tgcccaagaa gaagaggaag gttcccggga tggccgtgaa
      121 gagcatgaag gtgaagctgc gcctggacaa catgcccgag atccgcgccg gcctgtggaa
      181 gctgcacacc gaggtgaacg ccggcgtgcg ctactacacc gagtggctga gcctgctgcg
      241 ccaggagaac ctgtaccgcc gcagccccaa cggcgacggc gagcaggagt gctacaagac
      301 cgccgaggag tgcaaggccg agctgctgga gcgcctgcgc gcccgccagg tggagaacgg
      361 ccactgcggc cccgccggca gcgacgacga gctgctgcag ctggcccgcc agctgtacga
      421 gctgctggtg ccccaggcca tcggcgccaa gggcgacgcc cagcagatcg cccgcaagtt
      481 cctgagcccc ctggccgaca aggacgccgt gggcggcctg ggcatcgcca aggccggcaa
      541 caagccccgc tgggtgcgca tgcgcgaggc cggcgagccc ggctgggagg aggagaaggc
      601 caaggccgag gcccgcaaga gcaccgaccg caccgccgac gtgctgcgcg ccctggccga
      661 cttcggcctg aagcccctga tgcgcgtgta caccgacagc gacatgagca gcgtgcagtg
      721 gaagcccctg cgcaagggcc aggccgtgcg cacctgggac cgcgacatgt tccagcaggc
      781 catcgagcgc atgatgagct gggagagctg gaaccagcgc gtgggcgagg cctacgccaa
      841 gctggtggag cagaagagcc gcttcgagca gaagaacttc gtgggccagg agcacctggt
      901 gcagctggtg aaccagctgc agcaggacat gaaggaggcc agccacggcc tggagagcaa
      961 ggagcagacc gcccactacc tgaccggccg cgccctgcgc ggcagcgaca aggtgttcga
     1021 gaagtgggag aagctggacc ccgacgcccc cttcgacctg tacgacaccg agatcaagaa
     1081 cgtgcagcgc cgcaacaccc gccgcttcgg cagccacgac ctgttcgcca agctggccga
     1141 gcccaagtac caggccctgt ggcgcgagga cgccagcttc ctgacccgct acgccgtgta
     1201 caacagcatc gtgcgcaagc tgaaccacgc caagatgttc gccaccttca ccctgcccga
     1261 cgccaccgcc caccccatct ggacccgctt cgacaagctg ggcggcaacc tgcaccagta
     1321 caccttcctg ttcaacgagt tcggcgaggg ccgccacgcc atccgcttcc agaagctgct
     1381 gaccgtggag gacggcgtgg ccaaggaggt ggacgacgtg accgtgccca tcagcatgag
     1441 cgcccagctg gacgacctgc tgccccgcga cccccacgag ctggtggccc tgtacttcca
     1501 ggactacggc gccgagcagc acctggccgg cgagttcggc ggcgccaaga tccagtaccg
     1561 ccgcgaccag ctgaaccacc tgcacgcccg ccgcggcgcc cgcgacgtgt acctgaacct
     1621 gagcgtgcgc gtgcagagcc agagcgaggc ccgcggcgag cgccgccccc cctacgccgc
     1681 cgtgttccgc ctggtgggcg acaaccaccg cgccttcgtg cacttcgaca agctgagcga
     1741 ctacctggcc gagcaccccg acgacggcaa gctgggcagc gagggcctgc tgagcggcct
     1801 gcgcgtgatg agcgtggacc tgggcctgcg caccagcgcc agcatcagcg tgttccgcgt
     1861 ggcccgcaag gacgagctga agcccaacag cgagggccgc gtgcccttct gcttccccat
     1921 cgagggcaac gagaacctgg tggccgtgca cgagcgcagc cagctgctga agctgcccgg
     1981 cgagaccgag agcaaggacc tgcgcgccat ccgcgaggag cgccagcgca ccctgcgcca
     2041 gctgcgcacc cagctggcct acctgcgcct gctggtgcgc tgcggcagcg aggacgtggg
     2101 ccgccgcgag cgcagctggg ccaagctgat cgagcagccc atggacgcca accagatgac
     2161 ccccgactgg cgcgaggcct tcgaggacga gctgcagaag ctgaagagcc tgtacggcat
     2221 ctgcggcgac cgcgagtgga ccgaggccgt gtacgagagc gtgcgccgcg tgtggcgcca
     2281 catgggcaag caggtgcgcg actggcgcaa ggacgtgcgc agcggcgagc gccccaagat
     2341 ccgcggctac cagaaggacg tggtgggcgg caacagcatc gagcagatcg agtacctgga
     2401 gcgccagtac aagttcctga agagctggag cttcttcggc aaggtgagcg gccaggtgat
     2461 ccgcgccgag aagggcagcc gcttcgccat caccctgcgc gagcacatcg accacgccaa
     2521 ggaggaccgc ctgaagaagc tggccgaccg catcatcatg gaggccctgg gctacgtgta
     2581 cgccctggac gacgagcgcg gcaagggcaa gtgggtggcc aagtaccccc cctgccagct
     2641 gatcctgctg gaggagctga gcgagtacca gttcaacaac gaccgccccc ccagcgagaa
     2701 caaccagctg atgcagtgga gccaccgcgg cgtgttccag gagctgctga accaggccca
     2761 ggtgcacgac ctgctggtgg gcaccatgta cgccgccttc agcagccgct tcgacgcccg
     2821 caccggcgcc cccggcatcc gctgccgccg cgtgcccgcc cgctgcgccc gcgagcagaa
     2881 ccccgagccc ttcccctggt ggctgaacaa gttcgtggcc gagcacaagc tggacggctg
     2941 ccccctgcgc gccgacgacc tgatccccac cggcgagggc gagttcttcg tgagcccctt
     3001 cagcgccgag gagggcgact tccaccagat ccacgccgac ctgaacgccg cccagaacct
     3061 gcagcgccgc ctgtggagcg acttcgacat cagccagatc cgcctgcgct gcgactgggg
     3121 cgaggtggac ggcgagcccg tgctgatccc ccgcaccacc ggcaagcgca ccgccgacag
     3181 ctacggcaac aaggtgttct acaccaagac cggcgtgacc tactacgagc gcgagcgcgg
     3241 caagaagcgc cgcaaggtgt tcgcccagga ggagctgagc gaggaggagg ccgagctgct
     3301 ggtggaggcc gacgaggccc gcgagaagag cgtggtgctg atgcgcgacc ccagcggcat
     3361 catcaaccgc ggcgactgga cccgccagaa ggagttctgg agcatggtga accagcgcat
     3421 cgagggctac ctggtgaagc agatccgcag ccgcgtgcgc ctgcaggaga gcgcctgcga
     3481 gaacaccggc gacatcgcta gtcatcacca tcaccaccat catcaccatc actaggcggc
     3541 cgcataatgc ttaagtcgaa cagaaagtaa tcgtattgta cacggccgca taatcgaaat
     3601 taatacgact cactataggg aattcggtac ctgagaataa ctagcataac cccttggggc
     3661 ctctaaacgg gtcttgaggg gttttttgct gaaacctcag gcatttgaga agcacacggt
     3721 cacactgctt ccggtagtca ataaaccggt aaaccagcaa tagacataag cggctattta
     3781 acgaccctgc cctgaaccga cgaccgggtc gaatttgctt tcgaatttct gccattcatc
     3841 cgcttattat cacttattca ggcgtagcaa ccaggcgttt aagggcacca ataactgcct
     3901 taaaaaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt
     3961 ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc
     4021 accttgtcgc cttgcgtata atatttgccc atagtgaaaa cgggggcgaa gaagttgtcc
     4081 atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa
     4141 aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca
     4201 tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat
     4261 gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc
     4321 accagctcac cgtctttcat tgccatacgg aactccggat gagcattcat caggcgggca
     4381 agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag
     4441 gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc
     4501 tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt
     4561 ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt
     4621 agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat
     4681 tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat
     4741 tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct
     4801 gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg cttctgtttc
     4861 tatcagctgt ccctcctgtt cagctactga cggggtggtg cgtaacggca aaagcaccgc
     4921 cggacatcag cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc
     4981 agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt
     5041 gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
     5101 ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac
     5161 ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga
     5221 caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag
     5281 ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt
     5341 taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt
     5401 ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc
     5461 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac
     5521 cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt
     5581 taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg
     5641 ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg
     5701 ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat
     5761 cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     5821 agccccatac gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg
     5881 agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga
     5941 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     6001 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc
     6061 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
     6121 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
     6181 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
     6241 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
     6301 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
     6361 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
     6421 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
     6481 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
     6541 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
     6601 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
     6661 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
     6721 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
     6781 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
     6841 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
     6901 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
     6961 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
     7021 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     7081 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     7141 ctctccctta tgcgactcct gcattaggaa attaatacga
//
