LOCUS       pBFC0984_clean_f        6362 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 6362)
  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..6362
                     /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    251..254
                     /label=BsaI OH
     misc_feature    255..280
                     /label=2xBsaI_stuffer
     misc_feature    complement(256..261)
                     /label=BsaI
     misc_feature    275..280
                     /label=BsaI
     terminator      281..647
                     /label=TrrnB terminator (BBa_K1893035)
                     /label=TrrnB
     misc_feature    282..285
                     /label=BsaI OH
     terminator      581..626
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(665..1288)
                     /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        1307..1362
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1343..1361
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1380..1391
                     /label=RBS (strong)
     misc_feature    1395..1398
                     /label=BsmBI_OH
     CDS             1401..4304
                     /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    2115..2117
                     /label=R>A
     misc_feature    2130..2132
                     /label=H>A
     misc_feature    3972..3974
                     /label=R>A
     misc_feature    3987..3989
                     /label=H>A
     misc_feature    4308..4311
                     /label=BsaI Junction Scar
     terminator      4312..4368
                     /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      4312..4368
                     /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    4369..4372
                     /label=BsaI
     misc_feature    4373..4397
                     /label=ConE scar
     protein_bind    4373..4376
                     /label=BsmBI
     terminator      complement(4417..4473)
                     /label=ECK120033737 Terminator (Voigt)
                     /note="rho-independent terminator of thrLABC operon"
     terminator      complement(4417..4473)
                     /label=ECK120033737 Terminator (Voigt)
                     /note="rho-independent terminator of thrLABC operon"
     misc_feature    complement(4474..4582)
                     /label=CamR Terminator
     misc_feature    complement(4474..4582)
                     /label=CamR Terminator
     CDS             complement(4583..5242)
                     /label=CamR
     misc_feature    5009..5012
                     /label=BbsI OH
     promoter        complement(5243..5347)
                     /label=CamR Promoter
     promoter        complement(5243..5347)
                     /label=CamR Promoter
     rep_origin      complement(5615..6159)
                     /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(5615..6159)
                     /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      6260..6349
                     /label=ECK120029600 Terminator (Voigt)
                     /note="rho-independent terminator of spheroplast protein Y 
                     transcript"
     terminator      6260..6349
                     /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 aaacagagac ctcgtttacc tatcggtctc atgcttgggc ccgaacaaaa
      301 actcatctca gaagaggatc tgaatagcgc cgtcgaccat catcatcatc atcattgagt
      361 ttaaacgctc tccagcttgg gtgttttggc ggatgagaga agattttcag cctgatacag
      421 attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg
      481 gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt
      541 gtggggtctg cccatgcgag agtaggcaac tgccaggcat caaataaaac gaaaggctca
      601 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaactcgc cagatatcga
      661 cgtcttaaga cccactttca catttaagtt gtttttctaa tccgcatatg atcaattcaa
      721 ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa taattcgata gcttgtcgta
      781 ataatggcgg catactatca gtagtaggtg tttccctttc ttctttagcg acttgatgct
      841 cttgatcttc caatacgcaa cctaaagtaa aatgccccac agcgctgagt gcatataatg
      901 cattctctag tgaaaaacct tgttggcata aaaaggctaa ttgattttcg agagtttcat
      961 actgtttttc tgtaggccgt gtacctaaat gtacttttgc tccatcgcga tgacttagta
     1021 aagcacatct aaaactttta gcgttattac gtaaaaaatc ttgccagctt tccccttcta
     1081 aagggcaaaa gtgagtatgg tgcctatcta acatctcaat ggctaaggcg tcgagcaaag
     1141 cccgcttatt ttttacatgc caatacaatg taggctgctc tacacctagc ttctgggcga
     1201 gtttacgggt tgttaaacct tcgattccga cctcattaag cagctctaat gcgctgttaa
     1261 tcactttact tttatctaat ctagacatca ttaattccta atttttgttg acactctatc
     1321 gttgatagag ttattttacc actccctatc agtgatagag aaaagaattc aaaagatcta
     1381 aagaggagaa aggatcttac atgatcgaaa aaaaaaagtc cttcgccaag ggcatgggcg
     1441 tgaagtccac actcgtgtcc ggctccaaag tgtacatgac aaccttcgcc gaaggcagcg
     1501 acgccaggct ggaaaagatc gtggagggcg acagcatcag gagcgtgaat gagggcgagg
     1561 ccttcagcgc tgaaatggcc gataaaaacg ccggctataa gatcggcaac gccaaattca
     1621 gccatcctaa gggctacgcc gtggtggcta acaaccctct gtatacagga cccgtccagc
     1681 aggatatgct cggcctgaag gaaactctgg aaaagaggta cttcggcgag agcgctgatg
     1741 gcaatgacaa tatttgtatc caggtgatcc ataacatcct ggacattgaa aaaatcctcg
     1801 ccgaatacat taccaacgcc gcctacgccg tcaacaatat ctccggcctg gataaggaca
     1861 ttattggatt cggcaagttc tccacagtgt atacctacga cgaattcaaa gaccccgagc
     1921 accatagggc cgctttcaac aataacgata agctcatcaa cgccatcaag gcccagtatg
     1981 acgagttcga caacttcctc gataacccca gactcggcta tttcggccag gcctttttca
     2041 gcaaggaggg cagaaattac atcatcaatt acggcaacga atgctatgac attctggccc
     2101 tcctgagcgg actggcgcac tgggtggtcg ctaacaacga agaagagtcc aggatctcca
     2161 ggacctggct ctacaacctc gataagaacc tcgacaacga atacatctcc accctcaact
     2221 acctctacga caggatcacc aatgagctga ccaactcctt ctccaagaac tccgccgcca
     2281 acgtgaacta tattgccgaa actctgggaa tcaaccctgc cgaattcgcc gaacaatatt
     2341 tcagattcag cattatgaaa gagcagaaaa acctcggatt caatatcacc aagctcaggg
     2401 aagtgatgct ggacaggaag gatatgtccg agatcaggaa aaatcataag gtgttcgact
     2461 ccatcaggac caaggtctac accatgatgg actttgtgat ttataggtat tacatcgaag
     2521 aggatgccaa ggtggctgcc gccaataagt ccctccccga taatgagaag tccctgagcg
     2581 agaaggatat ctttgtgatt aacctgaggg gctccttcaa cgacgaccag aaggatgccc
     2641 tctactacga tgaagctaat agaatttgga gaaagctcga aaatatcatg cacaacatca
     2701 aggaatttag gggaaacaag acaagagagt ataagaagaa ggacgcccct agactgccca
     2761 gaatcctgcc cgctggccgt gatgtttccg ccttcagcaa actcatgtat gccctgacca
     2821 tgttcctgga tggcaaggag atcaacgacc tcctgaccac cctgattaat aaattcgata
     2881 acatccagag cttcctgaag gtgatgcctc tcatcggagt caacgctaag ttcgtggagg
     2941 aatacgcctt tttcaaagac tccgccaaga tcgccgatga gctgaggctg atcaagtcct
     3001 tcgctagaat gggagaacct attgccgatg ccaggagggc catgtatatc gacgccatcc
     3061 gtattttagg aaccaacctg tcctatgatg agctcaaggc cctcgccgac accttttccc
     3121 tggacgagaa cggaaacaag ctcaagaaag gcaagcacgg catgagaaat ttcattatta
     3181 ataacgtgat cagcaataaa aggttccact acctgatcag atacggtgat cctgcccacc
     3241 tccatgagat cgccaaaaac gaggccgtgg tgaagttcgt gctcggcagg atcgctgaca
     3301 tccagaaaaa acagggccag aacggcaaga accagatcga caggtactac gaaacttgta
     3361 tcggaaagga taagggcaag agcgtgagcg aaaaggtgga cgctctcaca aagatcatca
     3421 ccggaatgaa ctacgaccaa ttcgacaaga aaaggagcgt cattgaggac accggcaggg
     3481 aaaacgccga gagggagaag tttaaaaaga tcatcagcct gtacctcacc gtgatctacc
     3541 acatcctcaa gaatattgtc aatatcaacg ccaggtacgt catcggattc cattgcgtcg
     3601 agcgtgatgc tcaactgtac aaggagaaag gctacgacat caatctcaag aaactggaag
     3661 agaagggatt cagctccgtc accaagctct gcgctggcat tgatgaaact gcccccgata
     3721 agagaaagga cgtggaaaag gagatggctg aaagagccaa ggagagcatt gacagcctcg
     3781 agagcgccaa ccccaagctg tatgccaatt acatcaaata cagcgacgag aagaaagccg
     3841 aggagttcac caggcagatt aacagggaga aggccaaaac cgccctgaac gcctacctga
     3901 ggaacaccaa gtggaatgtg atcatcaggg aggacctcct gagaattgac aacaagacat
     3961 gtaccctgtt cgcaaacaag gccgtcgccc tggaagtggc caggtatgtc cacgcctata
     4021 tcaacgacat tgccgaggtc aattcctact tccaactgta ccattacatc atgcagagaa
     4081 ttatcatgaa tgagaggtac gagaaaagca gcggaaaggt gtccgagtac ttcgacgctg
     4141 tgaatgacga gaagaagtac aacgataggc tcctgaaact gctgtgtgtg cctttcggct
     4201 actgtatccc caggtttaag aacctgagca tcgaggccct gttcgatagg aacgaggccg
     4261 ccaagttcga caaggagaaa aagaaggtgt ccggcaattc ctaataaatc cctcggtacc
     4321 aaagacgaac aataagacgc tgaaaagcgt cttttttcgt tttggtccgc tgagcagtta
     4381 cagagatgtt acgaaccact agtgcactgc agtacacctt tggtcgaaaa aaaaagcccg
     4441 cactgtcagg tgcgggcttt tttctgtgtt tccaccaata aaaaacgccc ggcggcaacc
     4501 gagcgttctg aacaaatcca gatggagttc tgaggtcatt actggatcta tcaacaggag
     4561 tccaagcgag ctcgatatca aattacgccc cgccctgcca ctcatcgcag tactgttgta
     4621 attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg
     4681 ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg
     4741 cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat
     4801 tggctgaaac gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac
     4861 cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt
     4921 cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa
     4981 cactatccca tatcaccagc tcaccgtctt tcattgccat acgaaattcc ggatgagcat
     5041 tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta
     5101 cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa
     5161 ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat
     5221 atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc gataactcaa
     5281 aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc
     5341 cgatcaagcg ctagcggagt gtatactggc ttactatgtt ggcactgatg agggtgtcag
     5401 tgaagtgctt catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga
     5461 tacaggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg
     5521 cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt
     5581 aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca
     5641 agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat
     5701 accaggcgtt tcccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac
     5761 cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg
     5821 ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct
     5881 gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac
     5941 tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa
     6001 ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc
     6061 aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt
     6121 tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag
     6181 ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc
     6241 cccatacgat ataagttgtt tcagccaaaa aacttaagac cgccggtctt gtccactacc
     6301 ttgcagtaat gcggtggaca ggatcggcgg ttttcttttc tcttctcaac cctgaattcg
     6361 ca
//
