LOCUS       pBFC0993_clean_f        6367 bp DNA     circular SYN 11-DEC-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6367)
  AUTHORS   Doudna Lab
  TITLE     Direct Submission
  JOURNAL   Exported Dec 11, 2024 from SnapGene 7.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          225..250
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     source          251..254
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     misc_feature    7..31
                     /label=ConS scar
     protein_bind    7..10
                     /label=BsmBI
     misc_feature    32..39
                     /label=Not on PCR assembled transcript
     misc_feature    40..43
                     /label=BsaI Junction Scar
     terminator      44..104
                     /label=L3S2P21 Terminator (Voigt)
                     /note="Strongest synthetic promoter from Voigt's Nature 
                     Methods paper
                     Also used by Alex Nielsen as a dSpyCas9 sgRNA terminator in
                     his CRISPRi logic gates paper
                     "
     terminator      44..104
                     /label=L3S2P21 Terminator (Voigt)
                     /note="Strongest synthetic promoter from Voigt's Nature 
                     Methods paper
                     Also used by Alex Nielsen as a dSpyCas9 sgRNA terminator in
                     his CRISPRi logic gates paper
                     "
     protein_bind    105..108
                     /label=BsaI
     misc_feature    109..133
                     /label=Con1 scar
     protein_bind    109..112
                     /label=BsmBI
     misc_feature    134..137
                     /label=BsaI Pt2 OH
     terminator      138..181
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     promoter        190..224
                     /label=J23119
                     /label=J23119 (SpeI) promoter
     misc_feature    225..254
                     
     misc_feature    255..285
                     /label=Sp10 (Emeric's Qi coli RFP-targeting pos control)
     terminator      286..652
                     /label=TrrnB terminator (BBa_K1893035)
                     /label=TrrnB
     terminator      586..631
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(670..1293)
                     /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        1312..1367
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1348..1366
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1385..1396
                     /label=RBS (strong)
     misc_feature    1400..1403
                     /label=BsmBI_OH
     CDS             1406..4309
                     /codon_start=1
                     /label=ddCasRx
                     /translation="MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDS
                     IRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLE
                     KRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTV
                     YTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYII
                     NYGNECYDILALLSGLAHWVVANNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITN
                     ELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRK
                     DMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFV
                     INLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPA
                     GRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAF
                     FKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDE
                     NGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQ
                     KKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRE
                     NAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLE
                     EKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKK
                     AEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFANKAVALEVARYVH
                     AYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCV
                     PFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     misc_feature    2120..2122
                     /label=R>A
     misc_feature    2135..2137
                     /label=H>A
     misc_feature    3977..3979
                     /label=R>A
     misc_feature    3992..3994
                     /label=H>A
     misc_feature    4313..4316
                     /label=BsaI Junction Scar
     terminator      4317..4373
                     /label=L3S2P55 Terminator (Voigt)
                     /note="A strong synthetic promoter from Voigt's Nature 
                     Methods paper
                     Also used by Alex Nielsen as a dSpyCas9 sgRNA terminator in
                     his CRISPRi logic gates paper
                     "
     terminator      4317..4373
                     /label=L3S2P55 Terminator (Voigt)
                     /note="A strong synthetic promoter from Voigt's Nature 
                     Methods paper
                     Also used by Alex Nielsen as a dSpyCas9 sgRNA terminator in
                     his CRISPRi logic gates paper
                     "
     protein_bind    4374..4377
                     /label=BsaI
     misc_feature    4378..4402
                     /label=ConE scar
     protein_bind    4378..4381
                     /label=BsmBI
     terminator      complement(4422..4478)
                     /label=ECK120033737 Terminator (Voigt)
                     /note="rho-independent terminator of thrLABC operon"
     terminator      complement(4422..4478)
                     /label=ECK120033737 Terminator (Voigt)
                     /note="rho-independent terminator of thrLABC operon"
     misc_feature    complement(4479..4587)
                     /label=CamR Terminator
     misc_feature    complement(4479..4587)
                     /label=CamR Terminator
     CDS             complement(4588..5247)
                     /label=CamR
     misc_feature    5014..5017
                     /label=BbsI OH
     promoter        complement(5248..5352)
                     /label=CamR Promoter
     promoter        complement(5248..5352)
                     /label=CamR Promoter
     rep_origin      complement(5620..6164)
                     /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.
                     "
     rep_origin      complement(5620..6164)
                     /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      6265..6354
                     /label=ECK120029600 Terminator (Voigt)
                     /note="rho-independent terminator of spheroplast protein Y 
                     transcript"
     terminator      6265..6354
                     /label=ECK120029600 Terminator (Voigt)
                     /note="rho-independent terminator of spheroplast protein Y 
                     transcript"
ORIGIN
        1 tctagactga actggccgat aattgcagac ggtttaaaca tccctcggta ccaaattcca
       61 gaaaagaggc ctcccgaaag gggggccttt tttcgttttg gtccgctgcc aaaccagatg
      121 tcaacacagc tacaacgatt tcagataaaa aaaatcctta gctttcgcta aggatgattt
      181 ctggaattct tgacagctag ctcagtccta ggtataatac tagtaacccc taccaactgg
      241 tcggggtttg aaacaactct ttgataacgt cttcgctact cgccaatgct tgggcccgaa
      301 caaaaactca tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat
      361 tgagtttaaa cgctctccag cttgggtgtt ttggcggatg agagaagatt ttcagcctga
      421 tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta
      481 gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg
      541 gtagtgtggg gtctgcccat gcgagagtag gcaactgcca ggcatcaaat aaaacgaaag
      601 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa ctcgccagat
      661 atcgacgtct taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa
      721 ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg
      781 tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg
      841 atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata
      901 taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt
      961 ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact
     1021 tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc
     1081 ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag
     1141 caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg
     1201 ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct
     1261 gttaatcact ttacttttat ctaatctaga catcattaat tcctaatttt tgttgacact
     1321 ctatcgttga tagagttatt ttaccactcc ctatcagtga tagagaaaag aattcaaaag
     1381 atctaaagag gagaaaggat cttacatgat cgaaaaaaaa aagtccttcg ccaagggcat
     1441 gggcgtgaag tccacactcg tgtccggctc caaagtgtac atgacaacct tcgccgaagg
     1501 cagcgacgcc aggctggaaa agatcgtgga gggcgacagc atcaggagcg tgaatgaggg
     1561 cgaggccttc agcgctgaaa tggccgataa aaacgccggc tataagatcg gcaacgccaa
     1621 attcagccat cctaagggct acgccgtggt ggctaacaac cctctgtata caggacccgt
     1681 ccagcaggat atgctcggcc tgaaggaaac tctggaaaag aggtacttcg gcgagagcgc
     1741 tgatggcaat gacaatattt gtatccaggt gatccataac atcctggaca ttgaaaaaat
     1801 cctcgccgaa tacattacca acgccgccta cgccgtcaac aatatctccg gcctggataa
     1861 ggacattatt ggattcggca agttctccac agtgtatacc tacgacgaat tcaaagaccc
     1921 cgagcaccat agggccgctt tcaacaataa cgataagctc atcaacgcca tcaaggccca
     1981 gtatgacgag ttcgacaact tcctcgataa ccccagactc ggctatttcg gccaggcctt
     2041 tttcagcaag gagggcagaa attacatcat caattacggc aacgaatgct atgacattct
     2101 ggccctcctg agcggactgg cgcactgggt ggtcgctaac aacgaagaag agtccaggat
     2161 ctccaggacc tggctctaca acctcgataa gaacctcgac aacgaataca tctccaccct
     2221 caactacctc tacgacagga tcaccaatga gctgaccaac tccttctcca agaactccgc
     2281 cgccaacgtg aactatattg ccgaaactct gggaatcaac cctgccgaat tcgccgaaca
     2341 atatttcaga ttcagcatta tgaaagagca gaaaaacctc ggattcaata tcaccaagct
     2401 cagggaagtg atgctggaca ggaaggatat gtccgagatc aggaaaaatc ataaggtgtt
     2461 cgactccatc aggaccaagg tctacaccat gatggacttt gtgatttata ggtattacat
     2521 cgaagaggat gccaaggtgg ctgccgccaa taagtccctc cccgataatg agaagtccct
     2581 gagcgagaag gatatctttg tgattaacct gaggggctcc ttcaacgacg accagaagga
     2641 tgccctctac tacgatgaag ctaatagaat ttggagaaag ctcgaaaata tcatgcacaa
     2701 catcaaggaa tttaggggaa acaagacaag agagtataag aagaaggacg cccctagact
     2761 gcccagaatc ctgcccgctg gccgtgatgt ttccgccttc agcaaactca tgtatgccct
     2821 gaccatgttc ctggatggca aggagatcaa cgacctcctg accaccctga ttaataaatt
     2881 cgataacatc cagagcttcc tgaaggtgat gcctctcatc ggagtcaacg ctaagttcgt
     2941 ggaggaatac gcctttttca aagactccgc caagatcgcc gatgagctga ggctgatcaa
     3001 gtccttcgct agaatgggag aacctattgc cgatgccagg agggccatgt atatcgacgc
     3061 catccgtatt ttaggaacca acctgtccta tgatgagctc aaggccctcg ccgacacctt
     3121 ttccctggac gagaacggaa acaagctcaa gaaaggcaag cacggcatga gaaatttcat
     3181 tattaataac gtgatcagca ataaaaggtt ccactacctg atcagatacg gtgatcctgc
     3241 ccacctccat gagatcgcca aaaacgaggc cgtggtgaag ttcgtgctcg gcaggatcgc
     3301 tgacatccag aaaaaacagg gccagaacgg caagaaccag atcgacaggt actacgaaac
     3361 ttgtatcgga aaggataagg gcaagagcgt gagcgaaaag gtggacgctc tcacaaagat
     3421 catcaccgga atgaactacg accaattcga caagaaaagg agcgtcattg aggacaccgg
     3481 cagggaaaac gccgagaggg agaagtttaa aaagatcatc agcctgtacc tcaccgtgat
     3541 ctaccacatc ctcaagaata ttgtcaatat caacgccagg tacgtcatcg gattccattg
     3601 cgtcgagcgt gatgctcaac tgtacaagga gaaaggctac gacatcaatc tcaagaaact
     3661 ggaagagaag ggattcagct ccgtcaccaa gctctgcgct ggcattgatg aaactgcccc
     3721 cgataagaga aaggacgtgg aaaaggagat ggctgaaaga gccaaggaga gcattgacag
     3781 cctcgagagc gccaacccca agctgtatgc caattacatc aaatacagcg acgagaagaa
     3841 agccgaggag ttcaccaggc agattaacag ggagaaggcc aaaaccgccc tgaacgccta
     3901 cctgaggaac accaagtgga atgtgatcat cagggaggac ctcctgagaa ttgacaacaa
     3961 gacatgtacc ctgttcgcaa acaaggccgt cgccctggaa gtggccaggt atgtccacgc
     4021 ctatatcaac gacattgccg aggtcaattc ctacttccaa ctgtaccatt acatcatgca
     4081 gagaattatc atgaatgaga ggtacgagaa aagcagcgga aaggtgtccg agtacttcga
     4141 cgctgtgaat gacgagaaga agtacaacga taggctcctg aaactgctgt gtgtgccttt
     4201 cggctactgt atccccaggt ttaagaacct gagcatcgag gccctgttcg ataggaacga
     4261 ggccgccaag ttcgacaagg agaaaaagaa ggtgtccggc aattcctaat aaatccctcg
     4321 gtaccaaaga cgaacaataa gacgctgaaa agcgtctttt ttcgttttgg tccgctgagc
     4381 agttacagag atgttacgaa ccactagtgc actgcagtac acctttggtc gaaaaaaaaa
     4441 gcccgcactg tcaggtgcgg gcttttttct gtgtttccac caataaaaaa cgcccggcgg
     4501 caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac
     4561 aggagtccaa gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg
     4621 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg
     4681 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac
     4741 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca
     4801 gggattggct gaaacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
     4861 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg
     4921 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg
     4981 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg
     5041 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt
     5101 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg
     5161 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt
     5221 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa
     5281 ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac
     5341 gtgcccgatc aagcgctagc ggagtgtata ctggcttact atgttggcac tgatgagggt
     5401 gtcagtgaag tgcttcatgt ggcaggagaa aaaaggctgc accggtgcgt cagcagaata
     5461 tgtgatacag gatatattcc gcttcctcgc tcactgactc gctacgctcg gtcgttcgac
     5521 tgcggcgagc ggaaatggct tacgaacggg gcggagattt cctggaagat gccaggaaga
     5581 tacttaacag ggaagtgaga gggccgcggc aaagccgttt ttccataggc tccgcccccc
     5641 tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga caggactata
     5701 aagataccag gcgtttcccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg
     5761 tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag
     5821 ttccgggtag gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga
     5881 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc
     5941 accactggca gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg
     6001 gttaaggcta aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc
     6061 ggttcaaaga gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt
     6121 cgttttcaga gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta
     6181 atcagataaa atatttctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag
     6241 tcagccccat acgatataag ttgtttcagc caaaaaactt aagaccgccg gtcttgtcca
     6301 ctaccttgca gtaatgcggt ggacaggatc ggcggttttc ttttctcttc tcaaccctga
     6361 attcgca
//
