LOCUS       Exported                5716 bp ds-DNA     circular SYN 04-FEB-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pPBO.ACT001
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5716)
  AUTHORS   Doudna Lab
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Feb 4, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5716
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(7..630)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     promoter        649..704
                     /gene="tetR"
                     /label=pTet
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    685..703
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             722..733
                     /label=RBS (strong)
     CDS             743..3643
                     /codon_start=1
                     /label=dCasRx
                     /translation="MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDS
                     IRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLE
                     KRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTV
                     YTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYII
                     NYGNECYDILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITN
                     ELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRK
                     DMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFV
                     INLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPA
                     GRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAF
                     FKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDE
                     NGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQ
                     KKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRE
                     NAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLE
                     EKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKK
                     AEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVH
                     AYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCV
                     PFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     misc_feature    1016..1046
                     /label=Start of HEPN1
     CDS             3650..3673
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3674..3697
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     terminator      3725..3841
                     /gene="Escherichia coli rrnB"
                     /label=TrrnB
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    3728..3772
                     /label=Terminator
     terminator      3812..3839
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     rep_origin      complement(4001..4546)
                     /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.
                     "
     terminator      4660..4754
                     /label=tLambda
                     /note="transcription terminator from phage lambda"
     CDS             complement(4775..5590)
                     /label=KanR
     promoter        complement(5591..5693)
                     /label=pCat
                     /note="promoter of the E. coli cat gene"
ORIGIN
        1 gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc
       61 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg
      121 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg
      181 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa
      241 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc
      301 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag
      361 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc
      421 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa
      481 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc
      541 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt
      601 aatcacttta cttttatcta atctagacat cattaattcc taatttttgt tgacactcta
      661 tcgttgatag agttatttta ccactcccta tcagtgatag agaaaagaat tcaaaagatc
      721 taaagaggag aaaggatctt acatgatcga aaaaaaaaag tccttcgcca agggcatggg
      781 cgtgaagtcc acactcgtgt ccggctccaa agtgtacatg acaaccttcg ccgaaggcag
      841 cgacgccagg ctggaaaaga tcgtggaggg cgacagcatc aggagcgtga atgagggcga
      901 ggccttcagc gctgaaatgg ccgataaaaa cgccggctat aagatcggca acgccaaatt
      961 cagccatcct aagggctacg ccgtggtggc taacaaccct ctgtatacag gacccgtcca
     1021 gcaggatatg ctcggcctga aggaaactct ggaaaagagg tacttcggcg agagcgctga
     1081 tggcaatgac aatatttgta tccaggtgat ccataacatc ctggacattg aaaaaatcct
     1141 cgccgaatac attaccaacg ccgcctacgc cgtcaacaat atctccggcc tggataagga
     1201 cattattgga ttcggcaagt tctccacagt gtatacctac gacgaattca aagaccccga
     1261 gcaccatagg gccgctttca acaataacga taagctcatc aacgccatca aggcccagta
     1321 tgacgagttc gacaacttcc tcgataaccc cagactcggc tatttcggcc aggccttttt
     1381 cagcaaggag ggcagaaatt acatcatcaa ttacggcaac gaatgctatg acattctggc
     1441 cctcctgagc ggactggcgc actgggtggt cgctaacaac gaagaagagt ccaggatctc
     1501 caggacctgg ctctacaacc tcgataagaa cctcgacaac gaatacatct ccaccctcaa
     1561 ctacctctac gacaggatca ccaatgagct gaccaactcc ttctccaaga actccgccgc
     1621 caacgtgaac tatattgccg aaactctggg aatcaaccct gccgaattcg ccgaacaata
     1681 tttcagattc agcattatga aagagcagaa aaacctcgga ttcaatatca ccaagctcag
     1741 ggaagtgatg ctggacagga aggatatgtc cgagatcagg aaaaatcata aggtgttcga
     1801 ctccatcagg accaaggtct acaccatgat ggactttgtg atttataggt attacatcga
     1861 agaggatgcc aaggtggctg ccgccaataa gtccctcccc gataatgaga agtccctgag
     1921 cgagaaggat atctttgtga ttaacctgag gggctccttc aacgacgacc agaaggatgc
     1981 cctctactac gatgaagcta atagaatttg gagaaagctc gaaaatatca tgcacaacat
     2041 caaggaattt aggggaaaca agacaagaga gtataagaag aaggacgccc ctagactgcc
     2101 cagaatcctg cccgctggcc gtgatgtttc cgccttcagc aaactcatgt atgccctgac
     2161 catgttcctg gatggcaagg agatcaacga cctcctgacc accctgatta ataaattcga
     2221 taacatccag agcttcctga aggtgatgcc tctcatcgga gtcaacgcta agttcgtgga
     2281 ggaatacgcc tttttcaaag actccgccaa gatcgccgat gagctgaggc tgatcaagtc
     2341 cttcgctaga atgggagaac ctattgccga tgccaggagg gccatgtata tcgacgccat
     2401 ccgtatttta ggaaccaacc tgtcctatga tgagctcaag gccctcgccg acaccttttc
     2461 cctggacgag aacggaaaca agctcaagaa aggcaagcac ggcatgagaa atttcattat
     2521 taataacgtg atcagcaata aaaggttcca ctacctgatc agatacggtg atcctgccca
     2581 cctccatgag atcgccaaaa acgaggccgt ggtgaagttc gtgctcggca ggatcgctga
     2641 catccagaaa aaacagggcc agaacggcaa gaaccagatc gacaggtact acgaaacttg
     2701 tatcggaaag gataagggca agagcgtgag cgaaaaggtg gacgctctca caaagatcat
     2761 caccggaatg aactacgacc aattcgacaa gaaaaggagc gtcattgagg acaccggcag
     2821 ggaaaacgcc gagagggaga agtttaaaaa gatcatcagc ctgtacctca ccgtgatcta
     2881 ccacatcctc aagaatattg tcaatatcaa cgccaggtac gtcatcggat tccattgcgt
     2941 cgagcgtgat gctcaactgt acaaggagaa aggctacgac atcaatctca agaaactgga
     3001 agagaaggga ttcagctccg tcaccaagct ctgcgctggc attgatgaaa ctgcccccga
     3061 taagagaaag gacgtggaaa aggagatggc tgaaagagcc aaggagagca ttgacagcct
     3121 cgagagcgcc aaccccaagc tgtatgccaa ttacatcaaa tacagcgacg agaagaaagc
     3181 cgaggagttc accaggcaga ttaacaggga gaaggccaaa accgccctga acgcctacct
     3241 gaggaacacc aagtggaatg tgatcatcag ggaggacctc ctgagaattg acaacaagac
     3301 atgtaccctg ttcgcaaaca aggccgtcgc cctggaagtg gccaggtatg tccacgccta
     3361 tatcaacgac attgccgagg tcaattccta cttccaactg taccattaca tcatgcagag
     3421 aattatcatg aatgagaggt acgagaaaag cagcggaaag gtgtccgagt acttcgacgc
     3481 tgtgaatgac gagaagaagt acaacgatag gctcctgaaa ctgctgtgtg tgcctttcgg
     3541 ctactgtatc cccaggttta agaacctgag catcgaggcc ctgttcgata ggaacgaggc
     3601 cgccaagttc gacaaggaga aaaagaaggt gtccggcaat tccctcgagg attataaaga
     3661 tgatgatgat aaagattata aagatgatga tgataaagct agctaataag gtctcatagg
     3721 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
     3781 gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt
     3841 ttatacctag ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga
     3901 ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag
     3961 atacttaaca gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc
     4021 ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat
     4081 aaagatacca ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc
     4141 ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc
     4201 agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc
     4261 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa
     4321 gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc
     4381 cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc
     4441 tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt
     4501 ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat
     4561 taatcagata aaatatttct agatttcagt gcaatttatc tcttcaaatg tagcacctga
     4621 agtcagcccc atacgatata agttgttact agtgcttgga ttctcaccaa taaaaaacgc
     4681 ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc
     4741 tatcaacagg agtccaagcg agctcgatat caaattagaa aaactcatcg agcatcaaat
     4801 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     4861 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     4921 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     4981 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     5041 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     5101 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     5161 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     5221 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     5281 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     5341 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     5401 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     5461 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     5521 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     5581 tatggctcat tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg
     5641 gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtttc
     5701 attttcgcca gatatc
//
