LOCUS       sequence-388294-        6367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RfxCas13d Negative Control (RFP targeting).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6367)
  AUTHORS   Adler BA, Hessler T, Cress BF, Lahiri A, Mutalik VK, Barrangou R, 
            Banfield J, Doudna JA
  TITLE     Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing.
  JOURNAL   Nat Microbiol. 2022 Dec;7(12):1967-1979. doi: 
            10.1038/s41564-022-01258-x. Epub 2022 Oct 31.
  PUBMED    36316451
REFERENCE   2  (bases 1 to 6367)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41564-022-01258-x"; journalName: "Nat Microbiol"; date: 
            "2022-12"; volume: "7"; issue: "12"; pages: "1967-1979"
COMMENT     Sequence Label: pBA630
FEATURES             Location/Qualifiers
     source          1..6367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(14..31)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(47..76)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(120..149)
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     promoter        complement(150..184)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     terminator      193..236
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     rep_origin      577..1122
                     /direction=RIGHT
                     /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.
                     "
     primer_bind     1023..1042
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1239..1256
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             1495..2154
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1561..1580)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2225..2259
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(2435..5335)
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDS
                     IRSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLE
                     KRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTV
                     YTYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYII
                     NYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITN
                     ELTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRK
                     DMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFV
                     INLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPA
                     GRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAF
                     FKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDE
                     NGNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQ
                     KKQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGRE
                     NAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLE
                     EKGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKK
                     AEEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVH
                     AYINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCV
                     PFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     RBS             5345..5356
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        5374..5429
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    5375..5393
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             5445..6071
                     /codon_start=1
                     /gene="tetR"
                     /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="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(5521..5540)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      complement(6109..6155)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     6308..6325
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
ORIGIN
        1 gcgtttaaac tcaatgatga tgatgatgat ggtcgacggc gctattcaga tcctcttctg
       61 agatgagttt ttgttcgggc ccaagcattg gcgagtagcg aagacgttat caaagagttg
      121 tttcaaaccc cgaccagttg gtaggggtta ctagtattat acctaggact gagctagctg
      181 tcaagaattc cagaaatcat ccttagcgaa agctaaggat tttttttatc tgaaatcgtt
      241 gtagctgtgt tgacatctgg tttggcagcg gaccaaaacg aaaaaaggcc cccctttcgg
      301 gaggcctctt ttctggaatt tggtaccgag ggatgtttaa accgtctgca attatcggcc
      361 agttcagtct agatgcgaat tcagggttga gaagagaaaa gaaaaccgcc gatcctgtcc
      421 accgcattac tgcaaggtag tggacaagac cggcggtctt aagttttttg gctgaaacaa
      481 cttatatcgt atggggctga cttcaggtgc tacatttgaa gagataaatt gcactgaaat
      541 ctagaaatat tttatctgat taataagatg atcttcttga gatcgttttg gtctgcgcgt
      601 aatctcttgc tctgaaaacg aaaaaaccgc cttgcagggc ggtttttcga aggttctctg
      661 agctaccaac tctttgaacc gaggtaactg gcttggagga gcgcagtcac caaaacttgt
      721 cctttcagtt tagccttaac cggcgcatga cttcaagact aactcctcta aatcaattac
      781 cagtggctgc tgccagtggt gcttttgcat gtctttccgg gttggactca agacgatagt
      841 taccggataa ggcgcagcgg tcggactgaa cggggggttc gtgcatacag tccagcttgg
      901 agcgaactgc ctacccggaa ctgagtgtca ggcgtggaat gagacaaacg cggccataac
      961 agcggaatga caccggtaaa ccgaaaggca ggaacaggag agcgcacgag ggagccgcca
     1021 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccaccactg atttgagcgt
     1081 cagatttcgt gatgcttgtc aggggggcgg agcctatgga aaaacggctt tgccgcggcc
     1141 ctctcacttc cctgttaagt atcttcctgg catcttccag gaaatctccg ccccgttcgt
     1201 aagccatttc cgctcgccgc agtcgaacga ccgagcgtag cgagtcagtg agcgaggaag
     1261 cggaatatat cctgtatcac atattctgct gacgcaccgg tgcagccttt tttctcctgc
     1321 cacatgaagc acttcactga caccctcatc agtgccaaca tagtaagcca gtatacactc
     1381 cgctagcgct tgatcgggca cgtaagaggt tccaactttc accataatga aataagatca
     1441 ctaccgggcg tattttttga gttatcgaga ttttcaggag ctaaggaagc taaaatggag
     1501 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
     1561 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
     1621 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     1681 cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg caatgaaaga cggtgagctg
     1741 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
     1801 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     1861 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     1921 tttttcgttt cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     1981 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     2041 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
     2101 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttgat
     2161 atcgagctcg cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga
     2221 tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtggaaaca cagaaaaaag
     2281 cccgcacctg acagtgcggg cttttttttc gaccaaaggt gtactgcagt gcactagtgg
     2341 ttcgtaacat ctctgtaact gctcagcgga ccaaaacgaa aaaagacgct tttcagcgtc
     2401 ttattgttcg tctttggtac cgagggattt attaggaatt gccggacacc ttctttttct
     2461 ccttgtcgaa cttggcggcc tcgttcctat cgaacagggc ctcgatgctc aggttcttaa
     2521 acctggggat acagtagccg aaaggcacac acagcagttt caggagccta tcgttgtact
     2581 tcttctcgtc attcacagcg tcgaagtact cggacacctt tccgctgctt ttctcgtacc
     2641 tctcattcat gataattctc tgcatgatgt aatggtacag ttggaagtag gaattgacct
     2701 cggcaatgtc gttgatatag gcgtggacat acctggccac ttccaggtgg acggccttgt
     2761 ttctgaacag ggtacatgtc ttgttgtcaa ttctcaggag gtcctccctg atgatcacat
     2821 tccacttggt gttcctcagg taggcgttca gggcggtttt ggccttctcc ctgttaatct
     2881 gcctggtgaa ctcctcggct ttcttctcgt cgctgtattt gatgtaattg gcatacagct
     2941 tggggttggc gctctcgagg ctgtcaatgc tctccttggc tctttcagcc atctcctttt
     3001 ccacgtcctt tctcttatcg ggggcagttt catcaatgcc agcgcagagc ttggtgacgg
     3061 agctgaatcc cttctcttcc agtttcttga gattgatgtc gtagcctttc tccttgtaca
     3121 gttgagcatc acgctcgacg caatggaatc cgatgacgta cctggcgttg atattgacaa
     3181 tattcttgag gatgtggtag atcacggtga ggtacaggct gatgatcttt ttaaacttct
     3241 ccctctcggc gttttccctg ccggtgtcct caatgacgct ccttttcttg tcgaattggt
     3301 cgtagttcat tccggtgatg atctttgtga gagcgtccac cttttcgctc acgctcttgc
     3361 ccttatcctt tccgatacaa gtttcgtagt acctgtcgat ctggttcttg ccgttctggc
     3421 cctgtttttt ctggatgtca gcgatcctgc cgagcacgaa cttcaccacg gcctcgtttt
     3481 tggcgatctc atggaggtgg gcaggatcac cgtatctgat caggtagtgg aaccttttat
     3541 tgctgatcac gttattaata atgaaatttc tcatgccgtg cttgcctttc ttgagcttgt
     3601 ttccgttctc gtccagggaa aaggtgtcgg cgagggcctt gagctcatca taggacaggt
     3661 tggttcctaa aatacggatg gcgtcgatat acatggccct cctggcatcg gcaataggtt
     3721 ctcccattct agcgaaggac ttgatcagcc tcagctcatc ggcgatcttg gcggagtctt
     3781 tgaaaaaggc gtattcctcc acgaacttag cgttgactcc gatgagaggc atcaccttca
     3841 ggaagctctg gatgttatcg aatttattaa tcagggtggt caggaggtcg ttgatctcct
     3901 tgccatccag gaacatggtc agggcataca tgagtttgct gaaggcggaa acatcacggc
     3961 cagcgggcag gattctgggc agtctagggg cgtccttctt cttatactct cttgtcttgt
     4021 ttcccctaaa ttccttgatg ttgtgcatga tattttcgag ctttctccaa attctattag
     4081 cttcatcgta gtagagggca tccttctggt cgtcgttgaa ggagcccctc aggttaatca
     4141 caaagatatc cttctcgctc agggacttct cattatcggg gagggactta ttggcggcag
     4201 ccaccttggc atcctcttcg atgtaatacc tataaatcac aaagtccatc atggtgtaga
     4261 ccttggtcct gatggagtcg aacaccttat gatttttcct gatctcggac atatccttcc
     4321 tgtccagcat cacttccctg agcttggtga tattgaatcc gaggtttttc tgctctttca
     4381 taatgctgaa tctgaaatat tgttcggcga attcggcagg gttgattccc agagtttcgg
     4441 caatatagtt cacgttggcg gcggagttct tggagaagga gttggtcagc tcattggtga
     4501 tcctgtcgta gaggtagttg agggtggaga tgtattcgtt gtcgaggttc ttatcgaggt
     4561 tgtagagcca ggtcctggag atcctggact cttcttcgtt gttatggacc acccagtgcc
     4621 tcagtccgct caggagggcc agaatgtcat agcattcgtt gccgtaattg atgatgtaat
     4681 ttctgccctc cttgctgaaa aaggcctggc cgaaatagcc gagtctgggg ttatcgagga
     4741 agttgtcgaa ctcgtcatac tgggccttga tggcgttgat gagcttatcg ttattgttga
     4801 aagcggccct atggtgctcg gggtctttga attcgtcgta ggtatacact gtggagaact
     4861 tgccgaatcc aataatgtcc ttatccaggc cggagatatt gttgacggcg taggcggcgt
     4921 tggtaatgta ttcggcgagg attttttcaa tgtccaggat gttatggatc acctggatac
     4981 aaatattgtc attgccatca gcgctctcgc cgaagtacct cttttccaga gtttccttca
     5041 ggccgagcat atcctgctgg acgggtcctg tatacagagg gttgttagcc accacggcgt
     5101 agcccttagg atggctgaat ttggcgttgc cgatcttata gccggcgttt ttatcggcca
     5161 tttcagcgct gaaggcctcg ccctcattca cgctcctgat gctgtcgccc tccacgatct
     5221 tttccagcct ggcgtcgctg ccttcggcga aggttgtcat gtacactttg gagccggaca
     5281 cgagtgtgga cttcacgccc atgcccttgg cgaaggactt ttttttttcg atcatgtaag
     5341 atcctttctc ctctttagat cttttgaatt cttttctcta tcactgatag ggagtggtaa
     5401 aataactcta tcaacgatag agtgtcaaca aaaattagga attaatgatg tctagattag
     5461 ataaaagtaa agtgattaac agcgcattag agctgcttaa tgaggtcgga atcgaaggtt
     5521 taacaacccg taaactcgcc cagaagctag gtgtagagca gcctacattg tattggcatg
     5581 taaaaaataa gcgggctttg ctcgacgcct tagccattga gatgttagat aggcaccata
     5641 ctcacttttg ccctttagaa ggggaaagct ggcaagattt tttacgtaat aacgctaaaa
     5701 gttttagatg tgctttacta agtcatcgcg atggagcaaa agtacattta ggtacacggc
     5761 ctacagaaaa acagtatgaa actctcgaaa atcaattagc ctttttatgc caacaaggtt
     5821 tttcactaga gaatgcatta tatgcactca gcgctgtggg gcattttact ttaggttgcg
     5881 tattggaaga tcaagagcat caagtcgcta aagaagaaag ggaaacacct actactgata
     5941 gtatgccgcc attattacga caagctatcg aattatttga tcaccaaggt gcagagccag
     6001 ccttcttatt cggccttgaa ttgatcatat gcggattaga aaaacaactt aaatgtgaaa
     6061 gtgggtctta agacgtcgat atctggcgag ttcaccgaca aacaacagat aaaacgaaag
     6121 gcccagtctt tcgactgagc ctttcgtttt atttgatgcc tggcagttgc ctactctcgc
     6181 atgggcagac cccacactac catcggcgct acggcgtttc acttctgagt tcggcatggg
     6241 gtcaggtggg accaccgcgc tactgccgcc aggcaaattc tgttttatca gaccgcttct
     6301 gcgttctgat ttaatctgta tcaggctgaa aatcttctct catccgccaa aacacccaag
     6361 ctggaga
//
