LOCUS       Exported                5874 bp ds-DNA     circular SYN 23-JUL-2020
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5874)
  AUTHORS   Doudna Lab
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Jul 23, 2020 from SnapGene 5.0.4
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5874
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    1..4
                     /label=MCS-1
                     /note="multiple cloning site 1"
     promoter        50..68
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    69..93
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     RBS             122..127
                     /note="ribosome binding site"
     misc_feature    133..274
                     /label=MCS-2
                     /note="multiple cloning site 2"
     CDS             136..138
                     /codon_start=1
                     /product="start codon"
                     /label=ATG
                     /translation="M"
     CDS             202..246
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     terminator      298..345
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(575..1390)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(1391..1482)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1498..2247)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(2443..3525)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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(3526..3603)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        3649..3667
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3668..3692
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by 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)."
     RBS             3723..3728
                     /note="ribosome binding site"
     CDS             3736..5856
                     /codon_start=1
                     /product="dCasPhi-1, C-terminally His tagged, original 
                     sequence as found in huge phage"
                     /label=dcasPhi-1
                     /label=C2C11 hypothetical protein CDS
                     /note="/modified_by=User"
                     /translation="MADTPTLFTQFLRHHLPGQRFRKDILKQAGRILANKGEDATIAFL
                     RGKSEESPPDFQPPVKCPIIACSRPLTEWPIYQASVAIQGYVYGQSLAEFEASDPGCSK
                     DGLLGWFDKTGVCTDYFSVQGLNLIFQNARKRYIGVQTKVTNRNEKRHKKLKRINAKRI
                     AEGLPELTSDEPESALDETGHLIDPPGLNTNIYCYQQVSPKPLALSEVNQLPTAYAGYS
                     TSGDDPIQPMVTKDRLSISKGQPGYIPEHQRALLSQKKHRRMRGYGLKARALLVIVRIQ
                     DDWAVIDLRSLLRNAYWRRIVQTKEPSTITKLLKLVTGDPVLDATRMVATFTYKPGIVQ
                     VRSAKCLKNKQGSKLFSERYLNETVSVTSIALGSNNLVAVATYRLVNGNTPELLQRFTL
                     PSHLVKDFERYKQAHDTLEDSIQKTAVASLPQGQQTEIRMWSMYGFREAQERVCQELGL
                     ADGSIPWNVMTATSTILTDLFLARGGDPKKCMFTSEPKKKKNSKQVLYKIRDRAWAKMY
                     RTLLSKETREAWNKALWGLKRGSPDYARLSKRKEELARRCVNYTISTAEKRAQCGRTIV
                     ALEDLNIGFFHGRGKQEPGWVGLFTRKKENRWLMQALHKAFLELAHHRGYHVIEVNPAY
                     TSQTCPVCRHCDPDNRDQHNREAFHCIGCGFRGNADLDVATHNIAMVAITGESLKRARG
                     SVASKTPQPLAAE"
     variation       4846..4848
                     /label=D371A
                     /note="RuvCI asp to ala variation"
     misc_feature    5857..5874
                     /label=His6 tag
ORIGIN
        1 taagtcgaac agaaagtaat cgtattgtac acggccgcat aatcgaaatt aatacgactc
       61 actatagggg aattgtgagc ggataacaat tccccatctt agtatattag ttaagtataa
      121 gaaggagata tacatatggc agatctcaat tggatatcgg ccggccacgc gatcgctgac
      181 gtcggtaccc tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg
      241 gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta
      301 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca
      361 tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag
      421 acataagcgg ctatttaacg accctgccct gaaccgacga caagctgacg accgggtctc
      481 cgcaagtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
      541 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
      601 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
      661 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
      721 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
      781 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
      841 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
      901 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcggt
      961 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     1021 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     1081 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     1141 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     1201 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     1261 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     1321 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     1381 tatggctcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     1441 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggcatg cagcgctctt
     1501 ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggtgtcag
     1561 ctcactcaaa agcggtaata cggttatcca cagaatcagg ggataaagcc ggaaagaaca
     1621 tgtgagcaaa aagcaaagca ccggaagaag ccaacgccgc aggcgttttt ccataggctc
     1681 cgcccccctg acgagcatca caaaaatcga cgctcaagcc agaggtggcg aaacccgaca
     1741 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1801 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1861 catagctcac gctgttggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1921 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1981 tccaacccgg taagacacga cttatcgcca ctggcagcag ccattggtaa ctgatttaga
     2041 ggactttgtc ttgaagttat gcacctgtta aggctaaact gaaagaacag attttggtga
     2101 gtgcggtcct ccaacccact taccttggtt caaagagttg gtagctcagc gaaccttgag
     2161 aaaaccaccg ttggtagcgg tggtttttct ttatttatga gatgatgaat caatcggtct
     2221 atcaagtcaa cgaacagcta ttccgttact ctagatttca gtgcaattta tctcttcaaa
     2281 tgtagcacct gaagtcagcc ccatacgata taagttgtaa ttctcatgtt agtcatgccc
     2341 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     2401 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     2461 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2521 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     2581 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     2641 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     2701 atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc
     2761 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     2821 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     2881 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     2941 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     3001 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     3061 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     3121 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     3181 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     3241 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     3301 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     3361 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     3421 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     3481 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     3541 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     3601 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaaatta atacgactca
     3661 ctatagggga attgtgagcg gataacaatt cccctgtaga aataattttg tttaacttta
     3721 ataaggagat ataccatggc ggacacccca acactgttca cacagttcct ccgacaccat
     3781 ctaccagggc aacgctttcg taaggatatc ctaaagcaag ctggccgaat ccttgccaac
     3841 aaaggagaag acgccacgat tgcgttcttg agaggaaaga gcgaagaatc gccaccagat
     3901 tttcaaccgc ccgtgaagtg tcctatcata gcgtgcagta ggccgctcac ggaatggcca
     3961 atttatcaag cctcggtggc tatacaaggc tacgtttatg gacaaagcct tgccgagttt
     4021 gaggcttcgg atccaggatg tagcaaagat ggtttgcttg ggtggtttga taaaaccgga
     4081 gtctgcactg actacttctc cgtccaaggt ctaaacctga tcttccagaa tgctcgcaag
     4141 cgctatatcg gagtccagac gaaagtcaca aatcgcaacg aaaagcgcca caaaaagctc
     4201 aagcggatca acgctaagcg tatagccgaa ggattgcctg aactaacttc tgatgagcca
     4261 gagtcagcct tagatgaaac tgggcacttg attgatccac ccggcctaaa cactaacatc
     4321 tactgctacc aacaggtaag tcctaaacct ttagcactct ccgaagtgaa tcaattgcca
     4381 acggcttacg cagggtactc cacgagtgga gacgatccca tacaaccgat ggttacgaaa
     4441 gatcgtctta gtatttcaaa gggccagcca gggtatatcc cggagcacca gagagcatta
     4501 ctttcccaga aaaaacatcg ccgtatgcgt ggctatggcc tcaaggccag agcactgcta
     4561 gtgatcgtaa gaatccaaga tgactgggct gtaatcgact tacgttcgtt gctgcggaac
     4621 gcctactggc gtcgtattgt gcagacaaaa gagccaagta caatcacgaa gttactcaaa
     4681 ctagtgactg gggatcctgt actagacgcc acaaggatgg ttgcaacttt cacatataaa
     4741 ccaggcatag ttcaggtgcg ttcagcgaag tgcctcaaga acaagcaagg ttctaagctt
     4801 ttctcggagc gctacctcaa tgagacggta agcgttacta gcatcgccct agggagcaat
     4861 aacctggttg cggttgcgac ttatcgtctt gtcaacggca acacccctga gttgcttcaa
     4921 cggttcacct taccaagtca tttagtaaag gatttcgagc ggtacaagca agctcatgac
     4981 acactagaag actccatcca gaaaaccgcc gtagcgagct tgccgcaagg tcagcaaaca
     5041 gaaataagaa tgtggtccat gtacgggttt agagaggcac aggaacgtgt ttgccaagaa
     5101 ctcggattag ctgatggctc gatcccatgg aatgtcatga cagctacgtc aacgattctc
     5161 acggatctat ttctggcacg tggcggagat ccgaaaaagt gtatgttcac ttctgagcct
     5221 aagaagaaaa aaaactccaa acaggtcttg tacaagattc gagacagagc ctgggccaaa
     5281 atgtatcgga ctcttctgtc gaaagagacg cgagaagctt ggaacaaggc actgtggggg
     5341 ctcaagagag gctctcctga ttatgcacgc ttgtccaaac gtaaagaaga gctggctcgt
     5401 cgctgcgtca actatacgat tagcacggct gagaagagag cccaatgcgg tcggaccatc
     5461 gttgctctgg aggatctcaa cattggtttc ttccatggta gagggaaaca agaacctggg
     5521 tgggttggat tattcacgcg gaagaaggaa aatcgatggc tcatgcaggc tttgcataaa
     5581 gcgttcttag agcttgcaca ccatcgtggc taccacgtta tagaggtcaa tccagcctac
     5641 acgtcacaaa cgtgtccggt gtgtaggcat tgtgacccag acaaccgtga ccagcataat
     5701 cgagaagctt tccactgcat aggatgcggg ttccgtggta atgctgatct cgatgtggcg
     5761 acccacaaca tcgcgatggt tgcaattacc ggagaatcgc tcaagcgtgc gagaggctcg
     5821 gtggcgtcaa aaaccccgca gcctctcgcg gctgaacatc accatcacca tcac
//
