LOCUS       sequence-369172-        8262 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pTet-RfxCas13d crRNA entry vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8262)
  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 8262)
  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: pBA562
FEATURES             Location/Qualifiers
     source          1..8262
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     gap             954..1020
                     /estimated_length=67
     RBS             1642..1653
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        1671..1726
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    1672..1690
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1742..2368
                     /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"
     terminator      complement(2406..2452)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2605..2622
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(2678..2695)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2711..2740)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(2779..2808)
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     promoter        complement(2809..2843)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     terminator      2852..2895
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     rep_origin      3236..3781
                     /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     3682..3701
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3898..3915
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             4154..4813
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4220..4239)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      4884..4918
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(5095..7995)
                     /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             8005..8016
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        8034..8089
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    8035..8053
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 ctcgaagcgt gtacgtttaa ggtaattcag aattttatac ttttcgagat agcggaacac
       61 gttggcagag ttcagttgac gaaagatctt caatttcagt tttttctcgt taatctcatt
      121 ctgaaacatc ttctttgaga tttcggatgg cgcaatgttc ttgaacgcaa agatatcctt
      181 accttctaat tccaagttga agtggacaat accgtgacgg atggaagaaa tcgcctcgtc
      241 gatgttggcg aagaaatctt cgatttcatt cttgttgtcc atattgaaat cgtacgaata
      301 gaacattttt aagttctcct taacctcgtt cttcttattt tcattataga tcttatcaac
      361 ttctccggac acgtacttct cttcaccttt attgttctta actgttttgc cgcgcatacg
      421 cccagtaata tcattctcgt tctccgtttc aagaatgttg cgaagagaaa agtaggccac
      481 agatgacacc ccaatgatgt tgcgaagaaa cgcttcattc tgacggttgc gggcgatgaa
      541 atctgaagtg gcaatttcgc cgtcttgcaa ataataatta tactttccac aattacggac
      601 gtacgtgtca agtttgttta acagcttatt ttcgatcaat tttttcaagt tctggtattc
      661 aaagatacgc ttaattttgt cattcgagat attacttaag cgcttatata cgtagttctt
      721 cagcaactga ctcatttcga tttccacgaa atgacaaaac gcgtatttga tgttcttgtc
      781 gttcaactct tctttgtcta agtaatactt gtaaaatact tggctctttt tcaactcact
      841 catgtccggt accttctcga tcaactcttt catgttatta acattctgga tttcttcgta
      901 gatgattttt gcaaagtttt ccttgtcatt cttacgtcca ataatttcgt ggtnnnnnnn
      961 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
     1021 gtcctcttcc ttgtaaagct tatcaaaggc ttctttcaca ttgcttacat aagcgtcacg
     1081 tttcgctgac tcacgataat aatcgtaaat aatgttacgc tttgacttac cttcaacctt
     1141 ctcgatgtta ttctcattaa tcttttgata attcgcctta ttcttttcaa atgagtactt
     1201 caggctgttg attttgttca gtttcttctt aatgtcgtta cgaaaaactt ccaattcctc
     1261 cgagttcacg ttttcattca gatagatctt tttcaacacg gcgaagtttt tcttgtcacg
     1321 gacatcgctc tcaaggatgt cagtctcaga atactcacga tcaatgttct cctttttccc
     1381 attcttcaaa ctcaaggtat tgtctttaag atagaccatt ttgtttgaga agaatttctt
     1441 taatttccca atgcgttttt gattttcctt ggtttccgtg ctgctgggat tcttgatata
     1501 catgtcaagg cgcatattaa gcaacgccga cagacgttcg tctgtgcgat tttcttccga
     1561 ttctgacttc actaagcggc cttcggacgt gtacttctta tgcgaaatgc ctcctacctt
     1621 cgtcactttc atgtaagatc ctttctcctc tttagatctt ttgaattctt ttctctatca
     1681 ctgataggga gtggtaaaat aactctatca acgatagagt gtcaacaaaa attaggaatt
     1741 aatgatgtct agattagata aaagtaaagt gattaacagc gcattagagc tgcttaatga
     1801 ggtcggaatc gaaggtttaa caacccgtaa actcgcccag aagctaggtg tagagcagcc
     1861 tacattgtat tggcatgtaa aaaataagcg ggctttgctc gacgccttag ccattgagat
     1921 gttagatagg caccatactc acttttgccc tttagaaggg gaaagctggc aagatttttt
     1981 acgtaataac gctaaaagtt ttagatgtgc tttactaagt catcgcgatg gagcaaaagt
     2041 acatttaggt acacggccta cagaaaaaca gtatgaaact ctcgaaaatc aattagcctt
     2101 tttatgccaa caaggttttt cactagagaa tgcattatat gcactcagcg ctgtggggca
     2161 ttttacttta ggttgcgtat tggaagatca agagcatcaa gtcgctaaag aagaaaggga
     2221 aacacctact actgatagta tgccgccatt attacgacaa gctatcgaat tatttgatca
     2281 ccaaggtgca gagccagcct tcttattcgg ccttgaattg atcatatgcg gattagaaaa
     2341 acaacttaaa tgtgaaagtg ggtcttaaga cgtcgatatc tggcgagttc accgacaaac
     2401 aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctgg
     2461 cagttgccta ctctcgcatg ggcagacccc acactaccat cggcgctacg gcgtttcact
     2521 tctgagttcg gcatggggtc aggtgggacc accgcgctac tgccgccagg caaattctgt
     2581 tttatcagac cgcttctgcg ttctgattta atctgtatca ggctgaaaat cttctctcat
     2641 ccgccaaaac acccaagctg gagagcgttt aaactcaatg atgatgatga tgatggtcga
     2701 cggcgctatt cagatcctct tctgagatga gtttttgttc gggcccaagc atgagaccga
     2761 taggtaaacg aggtctctgt ttcaaacccc gaccagttgg taggggttac tagtattata
     2821 cctaggactg agctagctgt caagaattcc agaaatcatc cttagcgaaa gctaaggatt
     2881 ttttttatct gaaatcgttg tagctgtgtt gacatctggt ttggcagcgg accaaaacga
     2941 aaaaaggccc ccctttcggg aggcctcttt tctggaattt ggtaccgagg gatgtttaaa
     3001 ccgtctgcaa ttatcggcca gttcagtcta gatgcgaatt cagggttgag aagagaaaag
     3061 aaaaccgccg atcctgtcca ccgcattact gcaaggtagt ggacaagacc ggcggtctta
     3121 agttttttgg ctgaaacaac ttatatcgta tggggctgac ttcaggtgct acatttgaag
     3181 agataaattg cactgaaatc tagaaatatt ttatctgatt aataagatga tcttcttgag
     3241 atcgttttgg tctgcgcgta atctcttgct ctgaaaacga aaaaaccgcc ttgcagggcg
     3301 gtttttcgaa ggttctctga gctaccaact ctttgaaccg aggtaactgg cttggaggag
     3361 cgcagtcacc aaaacttgtc ctttcagttt agccttaacc ggcgcatgac ttcaagacta
     3421 actcctctaa atcaattacc agtggctgct gccagtggtg cttttgcatg tctttccggg
     3481 ttggactcaa gacgatagtt accggataag gcgcagcggt cggactgaac ggggggttcg
     3541 tgcatacagt ccagcttgga gcgaactgcc tacccggaac tgagtgtcag gcgtggaatg
     3601 agacaaacgc ggccataaca gcggaatgac accggtaaac cgaaaggcag gaacaggaga
     3661 gcgcacgagg gagccgccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3721 ccaccactga tttgagcgtc agatttcgtg atgcttgtca ggggggcgga gcctatggaa
     3781 aaacggcttt gccgcggccc tctcacttcc ctgttaagta tcttcctggc atcttccagg
     3841 aaatctccgc cccgttcgta agccatttcc gctcgccgca gtcgaacgac cgagcgtagc
     3901 gagtcagtga gcgaggaagc ggaatatatc ctgtatcaca tattctgctg acgcaccggt
     3961 gcagcctttt ttctcctgcc acatgaagca cttcactgac accctcatca gtgccaacat
     4021 agtaagccag tatacactcc gctagcgctt gatcgggcac gtaagaggtt ccaactttca
     4081 ccataatgaa ataagatcac taccgggcgt attttttgag ttatcgagat tttcaggagc
     4141 taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg
     4201 gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac
     4261 cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta
     4321 tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat ttcgtatggc
     4381 aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca
     4441 tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt
     4501 tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa
     4561 agggtttatt gagaatatgt ttttcgtttc agccaatccc tgggtgagtt tcaccagttt
     4621 tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata
     4681 ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg
     4741 tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca
     4801 gggcggggcg taatttgata tcgagctcgc ttggactcct gttgatagat ccagtaatga
     4861 cctcagaact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg
     4921 gtggaaacac agaaaaaagc ccgcacctga cagtgcgggc ttttttttnc gaccaaaggt
     4981 gtactgcagt gcactagtgg ttcgtaacat ctctgtaact gctcagcgga ccaaaacgaa
     5041 aaaagacgct tttcagcgtc ttattgttcg tctttggtac cgagggattt attaggaatt
     5101 gccggacacc ttctttttct ccttgtcgaa cttggcggcc tcgttcctat cgaacagggc
     5161 ctcgatgctc aggttcttaa acctggggat acagtagccg aaaggcacac acagcagttt
     5221 caggagccta tcgttgtact tcttctcgtc attcacagcg tcgaagtact cggacacctt
     5281 tccgctgctt ttctcgtacc tctcattcat gataattctc tgcatgatgt aatggtacag
     5341 ttggaagtag gaattgacct cggcaatgtc gttgatatag gcgtggacat acctggccac
     5401 ttccaggtgg acggccttgt ttctgaacag ggtacatgtc ttgttgtcaa ttctcaggag
     5461 gtcctccctg atgatcacat tccacttggt gttcctcagg taggcgttca gggcggtttt
     5521 ggccttctcc ctgttaatct gcctggtgaa ctcctcggct ttcttctcgt cgctgtattt
     5581 gatgtaattg gcatacagct tggggttggc gctctcgagg ctgtcaatgc tctccttggc
     5641 tctttcagcc atctcctttt ccacgtcctt tctcttatcg ggggcagttt catcaatgcc
     5701 agcgcagagc ttggtgacgg agctgaatcc cttctcttcc agtttcttga gattgatgtc
     5761 gtagcctttc tccttgtaca gttgagcatc acgctcgacg caatggaatc cgatgacgta
     5821 cctggcgttg atattgacaa tattcttgag gatgtggtag atcacggtga ggtacaggct
     5881 gatgatcttt ttaaacttct ccctctcggc gttttccctg ccggtgtcct caatgacgct
     5941 ccttttcttg tcgaattggt cgtagttcat tccggtgatg atctttgtga gagcgtccac
     6001 cttttcgctc acgctcttgc ccttatcctt tccgatacaa gtttcgtagt acctgtcgat
     6061 ctggttcttg ccgttctggc cctgtttttt ctggatgtca gcgatcctgc cgagcacgaa
     6121 cttcaccacg gcctcgtttt tggcgatctc atggaggtgg gcaggatcac cgtatctgat
     6181 caggtagtgg aaccttttat tgctgatcac gttattaata atgaaatttc tcatgccgtg
     6241 cttgcctttc ttgagcttgt ttccgttctc gtccagggaa aaggtgtcgg cgagggcctt
     6301 gagctcatca taggacaggt tggttcctaa aatacggatg gcgtcgatat acatggccct
     6361 cctggcatcg gcaataggtt ctcccattct agcgaaggac ttgatcagcc tcagctcatc
     6421 ggcgatcttg gcggagtctt tgaaaaaggc gtattcctcc acgaacttag cgttgactcc
     6481 gatgagaggc atcaccttca ggaagctctg gatgttatcg aatttattaa tcagggtggt
     6541 caggaggtcg ttgatctcct tgccatccag gaacatggtc agggcataca tgagtttgct
     6601 gaaggcggaa acatcacggc cagcgggcag gattctgggc agtctagggg cgtccttctt
     6661 cttatactct cttgtcttgt ttcccctaaa ttccttgatg ttgtgcatga tattttcgag
     6721 ctttctccaa attctattag cttcatcgta gtagagggca tccttctggt cgtcgttgaa
     6781 ggagcccctc aggttaatca caaagatatc cttctcgctc agggacttct cattatcggg
     6841 gagggactta ttggcggcag ccaccttggc atcctcttcg atgtaatacc tataaatcac
     6901 aaagtccatc atggtgtaga ccttggtcct gatggagtcg aacaccttat gatttttcct
     6961 gatctcggac atatccttcc tgtccagcat cacttccctg agcttggtga tattgaatcc
     7021 gaggtttttc tgctctttca taatgctgaa tctgaaatat tgttcggcga attcggcagg
     7081 gttgattccc agagtttcgg caatatagtt cacgttggcg gcggagttct tggagaagga
     7141 gttggtcagc tcattggtga tcctgtcgta gaggtagttg agggtggaga tgtattcgtt
     7201 gtcgaggttc ttatcgaggt tgtagagcca ggtcctggag atcctggact cttcttcgtt
     7261 gttatggacc acccagtgcc tcagtccgct caggagggcc agaatgtcat agcattcgtt
     7321 gccgtaattg atgatgtaat ttctgccctc cttgctgaaa aaggcctggc cgaaatagcc
     7381 gagtctgggg ttatcgagga agttgtcgaa ctcgtcatac tgggccttga tggcgttgat
     7441 gagcttatcg ttattgttga aagcggccct atggtgctcg gggtctttga attcgtcgta
     7501 ggtatacact gtggagaact tgccgaatcc aataatgtcc ttatccaggc cggagatatt
     7561 gttgacggcg taggcggcgt tggtaatgta ttcggcgagg attttttcaa tgtccaggat
     7621 gttatggatc acctggatac aaatattgtc attgccatca gcgctctcgc cgaagtacct
     7681 cttttccaga gtttccttca ggccgagcat atcctgctgg acgggtcctg tatacagagg
     7741 gttgttagcc accacggcgt agcccttagg atggctgaat ttggcgttgc cgatcttata
     7801 gccggcgttt ttatcggcca tttcagcgct gaaggcctcg ccctcattca cgctcctgat
     7861 gctgtcgccc tccacgatct tttccagcct ggcgtcgctg ccttcggcga aggttgtcat
     7921 gtacactttg gagccggaca cgagtgtgga cttcacgccc atgcccttgg cgaaggactt
     7981 ttttttttcg atcatgtaag atcctttctc ctctttagat cttttgaatt cttttctcta
     8041 tcactgatag ggagtggtaa aataactcta tcaacgatag agtgtcaaca aaaattagga
     8101 attaatgatg tctagattag ataaaagtaa agtgattaac agcgcattag agctgcttaa
     8161 tgaggtcgga atcgaaggtt taacaacccg taaactcgcc cagaagctag gtgtagagca
     8221 gcctacattg tattggcatg taaaaaataa gcgggctttg ct
//
