LOCUS       Exported                6024 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 6024)
  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..6024
                     /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..6006
                     /codon_start=1
                     /product="dCasPhi-2, C-terminally His tagged, codon 
                     optimized for expression in E coli"
                     /label=dcasPhi-2
                     /translation="MPKPAVESEFSKVLKKHFPGERFRSSYMKRGGKILAAQGEEAVVA
                     YLQGKSEEEPPNFQPPAKCHVVTKSRDFAEWPIMKASEAIQRYIYALSTTERAACKPGK
                     SSESHAAWFAATGVSNHGYSHVQGLNLIFDHTLGRYDGVLKKVQLRNEKARARLESINA
                     SRADEGLPEIKAEEEEVATNETGHLLQPPGINPSFYVYQTISPQAYRPRDEIVLPPEYA
                     GYVRDPNAPIPLGVVRNRCDIQKGCPGYIPEWQREAGTAISPKTGKAVTVPGLSPKKNK
                     RMRRYWRSEKEKAQDALLVTVRIGTDWVVIDVRGLLRNARWRTIAPKDISLNALLDLFT
                     GDPVIDVRRNIVTFTYTLDACGTYARKWTLKGKQTKATLDKLTATQTVALVAIALGQTN
                     PISAGISRVTQENGALQCEPLDRFTLPDDLLKDISAYRIAWDRNEEELRARSVEALPEA
                     QQAEVRALDGVSKETARTQLCADFGLDPKRLPWDKMSSNTTFISEALLSNSVSRDQVFF
                     TPAPKKGAKKKAPVEVMRKDRTWARAYKPRLSVEAQKLKNEALWALKRTSPEYLKLSRR
                     KEELCRRSINYVIEKTRRRTQCQIVIPVIEDLNVRFFHGSGKRLPGWDNFFTAKKENRW
                     FIQGLHKAFSDLRTHRSFYVFEVRPERTSITCPKCGHCEVGNRDGEAFQCLSCGKTCNA
                     DLDVATHNLTQVALTGKTMPKREEPRDAQGTAPARKTKKASKSKAPPAEREDQTPAQEP
                     SQTS"
     variation       4915..4917
                     /label=D394A
                     /note="dRuvCI asp to ala variation"
     misc_feature    6007..6024
                     /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 ataccatgcc aaaacccgcg gttgagagtg aattctcgaa agtgcttaag
     3781 aagcacttcc ccggggagcg cttccgttcc agttacatga aacgcggtgg caagatctta
     3841 gccgcccagg gagaagaggc cgtagttgca tacttacagg gaaaatcaga ggaggaaccg
     3901 cctaacttcc aacctcccgc gaaatgtcat gtggttacta aaagtcgcga ttttgccgaa
     3961 tggccgatta tgaaagccag cgaggctatt caacgctaca tctacgcgtt gtcaaccact
     4021 gagcgtgccg cgtgtaaacc agggaaaagc agcgaatcac atgctgcctg gtttgcagcg
     4081 actggcgtca gtaaccacgg gtattcacat gtccaagggc ttaacttaat ctttgaccat
     4141 actctggggc gttacgatgg ggttttgaag aaggttcaac tgcgtaatga aaaggcacgt
     4201 gcccgtttgg agtcaattaa tgcatctcgt gccgatgagg gtttacctga aatcaaggcg
     4261 gaagaggaag aggtagccac aaatgagaca gggcatttgc ttcaaccgcc aggcattaat
     4321 cccagcttct atgtttatca aacgatctca cctcaggctt atcgtccacg cgacgagatt
     4381 gttttgcccc ccgagtatgc agggtacgtg cgtgatccca acgcccccat tccgttagga
     4441 gtagtccgca accgttgtga tattcagaaa ggctgtccgg gctatattcc tgagtggcag
     4501 cgcgaggcag gtactgcgat ctctccgaag actgggaaag ccgttacagt tcctggcctt
     4561 tcccctaaga agaacaagcg tatgcgccgc tattggcgtt cggaaaaaga gaaggcacaa
     4621 gatgcactgt tggttaccgt ccgtatcggt actgactggg tagtgattga tgtgcgcgga
     4681 ttattgcgta atgcccgctg gcgcaccatt gctccaaagg acatctcatt aaacgcgctt
     4741 ttagacctgt tcactggaga cccagtgatt gatgtccgcc gcaacattgt aacttttacc
     4801 tacacgcttg atgcctgcgg tacctatgct cgtaaatgga cgttaaaagg aaagcagaca
     4861 aaggcaacac tggataaact gactgccacc caaacagtag cgctggtagc gatcgccttg
     4921 ggccagacga accctatttc agcgggaatc tcccgtgtta cgcaggaaaa cggggcgctt
     4981 caatgtgagc cgctggaccg ctttacgtta cctgacgact tgttgaaaga tatcagtgcg
     5041 taccgcatcg catgggaccg caacgaggag gaattacgtg cccgttcagt cgaagcgtta
     5101 cccgaagcac agcaggccga agtacgtgct ctggacggcg taagcaagga gaccgctcgt
     5161 actcaattat gtgctgattt cggccttgat cccaagcgtc tgccttggga caagatgtcc
     5221 tcaaacacta cattcatcag cgaagccctg ttgtcaaata gtgtatcgcg tgaccaggtt
     5281 ttctttaccc cagctcctaa aaaaggagcc aaaaaaaaag ctccggtgga ggttatgcgc
     5341 aaggaccgta cttgggcccg tgcttataaa ccacgtctgt ctgttgaagc ccagaaactg
     5401 aaaaacgaag ctctgtgggc ccttaaacgt acgagtcccg aataccttaa attaagccgt
     5461 cgtaaagagg agctttgccg tcgcagtatc aattacgtta tcgagaagac ccgccgtcgt
     5521 acccagtgtc aaatcgtgat cccggtgatt gaagatctga acgtccgctt tttccacgga
     5581 tctgggaaac gtctgcctgg ctgggataat ttctttacag cgaaaaaaga gaaccgttgg
     5641 ttcatccagg gccttcacaa ggctttttcc gacttacgca cacatcgcag tttctacgtt
     5701 ttcgaggtac gtccagagcg cacctccatt acctgcccca aatgcgggca ctgcgaggtt
     5761 gggaaccgtg atggcgaagc gttccaatgc ctttcctgtg gaaaaacatg taatgcagac
     5821 ttagacgtag ccactcacaa cctgacacag gttgccctta ccggaaaaac catgcctaaa
     5881 cgtgaagaac cacgcgatgc ccaggggacc gctccggcac gtaagaccaa aaaagcatca
     5941 aagtcgaagg ccccccctgc tgaacgcgag gaccagaccc cggcccaaga acctagccag
     6001 accagccatc accatcacca tcac
//
