LOCUS       sequence-373573-        6362 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 6362)
  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 6362)
  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..6362
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(23..40)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(157..702)
                     /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.
                     "
     primer_bind     complement(237..256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1043..1086)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     promoter        1095..1129
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        1130..1159
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d 
                     gRNA (Konermann et al., 2018)"
     CDS             1198..1227
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1243..1260
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1316..1333)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1486..1532
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1570..2196)
                     /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     2101..2120
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        2212..2267
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    2248..2266
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             2285..2296
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2306..5206
                     /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"
     terminator      complement(5382..5416)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(5487..6146)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6061..6080
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
       61 ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac
      121 ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga
      181 caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag
      241 ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt
      301 taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt
      361 ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc
      421 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac
      481 cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt
      541 taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg
      601 ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg
      661 ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat
      721 cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
      781 agccccatac gatataagtt gtttcagcca aaaaacttaa gaccgccggt cttgtccact
      841 accttgcagt aatgcggtgg acaggatcgg cggttttctt ttctcttctc aaccctgaat
      901 tcgcatctag actgaactgg ccgataattg cagacggttt aaacatccct cggtaccaaa
      961 ttccagaaaa gaggcctccc gaaagggggg ccttttttcg ttttggtccg ctgccaaacc
     1021 agatgtcaac acagctacaa cgatttcaga taaaaaaaat ccttagcttt cgctaaggat
     1081 gatttctgga attcttgaca gctagctcag tcctaggtat aatactagta acccctacca
     1141 actggtcggg gtttgaaaca gagacctcgt ttacctatcg gtctcatgct tgggcccgaa
     1201 caaaaactca tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat
     1261 tgagtttaaa cgctctccag cttgggtgtt ttggcggatg agagaagatt ttcagcctga
     1321 tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta
     1381 gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg
     1441 gtagtgtggg gtctgcccat gcgagagtag gcaactgcca ggcatcaaat aaaacgaaag
     1501 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa ctcgccagat
     1561 atcgacgtct taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa
     1621 ttcaaggccg aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg
     1681 tcgtaataat ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg
     1741 atgctcttga tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata
     1801 taatgcattc tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt
     1861 ttcatactgt ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact
     1921 tagtaaagca catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc
     1981 ttctaaaggg caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag
     2041 caaagcccgc ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg
     2101 ggcgagttta cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct
     2161 gttaatcact ttacttttat ctaatctaga catcattaat tcctaatttt tgttgacact
     2221 ctatcgttga tagagttatt ttaccactcc ctatcagtga tagagaaaag aattcaaaag
     2281 atctaaagag gagaaaggat cttacatgat cgaaaaaaaa aagtccttcg ccaagggcat
     2341 gggcgtgaag tccacactcg tgtccggctc caaagtgtac atgacaacct tcgccgaagg
     2401 cagcgacgcc aggctggaaa agatcgtgga gggcgacagc atcaggagcg tgaatgaggg
     2461 cgaggccttc agcgctgaaa tggccgataa aaacgccggc tataagatcg gcaacgccaa
     2521 attcagccat cctaagggct acgccgtggt ggctaacaac cctctgtata caggacccgt
     2581 ccagcaggat atgctcggcc tgaaggaaac tctggaaaag aggtacttcg gcgagagcgc
     2641 tgatggcaat gacaatattt gtatccaggt gatccataac atcctggaca ttgaaaaaat
     2701 cctcgccgaa tacattacca acgccgccta cgccgtcaac aatatctccg gcctggataa
     2761 ggacattatt ggattcggca agttctccac agtgtatacc tacgacgaat tcaaagaccc
     2821 cgagcaccat agggccgctt tcaacaataa cgataagctc atcaacgcca tcaaggccca
     2881 gtatgacgag ttcgacaact tcctcgataa ccccagactc ggctatttcg gccaggcctt
     2941 tttcagcaag gagggcagaa attacatcat caattacggc aacgaatgct atgacattct
     3001 ggccctcctg agcggactga ggcactgggt ggtccataac aacgaagaag agtccaggat
     3061 ctccaggacc tggctctaca acctcgataa gaacctcgac aacgaataca tctccaccct
     3121 caactacctc tacgacagga tcaccaatga gctgaccaac tccttctcca agaactccgc
     3181 cgccaacgtg aactatattg ccgaaactct gggaatcaac cctgccgaat tcgccgaaca
     3241 atatttcaga ttcagcatta tgaaagagca gaaaaacctc ggattcaata tcaccaagct
     3301 cagggaagtg atgctggaca ggaaggatat gtccgagatc aggaaaaatc ataaggtgtt
     3361 cgactccatc aggaccaagg tctacaccat gatggacttt gtgatttata ggtattacat
     3421 cgaagaggat gccaaggtgg ctgccgccaa taagtccctc cccgataatg agaagtccct
     3481 gagcgagaag gatatctttg tgattaacct gaggggctcc ttcaacgacg accagaagga
     3541 tgccctctac tacgatgaag ctaatagaat ttggagaaag ctcgaaaata tcatgcacaa
     3601 catcaaggaa tttaggggaa acaagacaag agagtataag aagaaggacg cccctagact
     3661 gcccagaatc ctgcccgctg gccgtgatgt ttccgccttc agcaaactca tgtatgccct
     3721 gaccatgttc ctggatggca aggagatcaa cgacctcctg accaccctga ttaataaatt
     3781 cgataacatc cagagcttcc tgaaggtgat gcctctcatc ggagtcaacg ctaagttcgt
     3841 ggaggaatac gcctttttca aagactccgc caagatcgcc gatgagctga ggctgatcaa
     3901 gtccttcgct agaatgggag aacctattgc cgatgccagg agggccatgt atatcgacgc
     3961 catccgtatt ttaggaacca acctgtccta tgatgagctc aaggccctcg ccgacacctt
     4021 ttccctggac gagaacggaa acaagctcaa gaaaggcaag cacggcatga gaaatttcat
     4081 tattaataac gtgatcagca ataaaaggtt ccactacctg atcagatacg gtgatcctgc
     4141 ccacctccat gagatcgcca aaaacgaggc cgtggtgaag ttcgtgctcg gcaggatcgc
     4201 tgacatccag aaaaaacagg gccagaacgg caagaaccag atcgacaggt actacgaaac
     4261 ttgtatcgga aaggataagg gcaagagcgt gagcgaaaag gtggacgctc tcacaaagat
     4321 catcaccgga atgaactacg accaattcga caagaaaagg agcgtcattg aggacaccgg
     4381 cagggaaaac gccgagaggg agaagtttaa aaagatcatc agcctgtacc tcaccgtgat
     4441 ctaccacatc ctcaagaata ttgtcaatat caacgccagg tacgtcatcg gattccattg
     4501 cgtcgagcgt gatgctcaac tgtacaagga gaaaggctac gacatcaatc tcaagaaact
     4561 ggaagagaag ggattcagct ccgtcaccaa gctctgcgct ggcattgatg aaactgcccc
     4621 cgataagaga aaggacgtgg aaaaggagat ggctgaaaga gccaaggaga gcattgacag
     4681 cctcgagagc gccaacccca agctgtatgc caattacatc aaatacagcg acgagaagaa
     4741 agccgaggag ttcaccaggc agattaacag ggagaaggcc aaaaccgccc tgaacgccta
     4801 cctgaggaac accaagtgga atgtgatcat cagggaggac ctcctgagaa ttgacaacaa
     4861 gacatgtacc ctgttcagaa acaaggccgt ccacctggaa gtggccaggt atgtccacgc
     4921 ctatatcaac gacattgccg aggtcaattc ctacttccaa ctgtaccatt acatcatgca
     4981 gagaattatc atgaatgaga ggtacgagaa aagcagcgga aaggtgtccg agtacttcga
     5041 cgctgtgaat gacgagaaga agtacaacga taggctcctg aaactgctgt gtgtgccttt
     5101 cggctactgt atccccaggt ttaagaacct gagcatcgag gccctgttcg ataggaacga
     5161 ggccgccaag ttcgacaagg agaaaaagaa ggtgtccggc aattcctaat aaatccctcg
     5221 gtaccaaaga cgaacaataa gacgctgaaa agcgtctttt ttcgttttgg tccgctgagc
     5281 agttacagag atgttacgaa ccactagtgc actgcagtac acctttggtc gaaaaaaaag
     5341 cccgcactgt caggtgcggg cttttttctg tgtttccacc aataaaaaac gcccggcggc
     5401 aaccgagcgt tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca
     5461 ggagtccaag cgagctcgat atcaaattac gccccgccct gccactcatc gcagtactgt
     5521 tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga
     5581 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg
     5641 ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag
     5701 ggattggctg aaacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt
     5761 tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg
     5821 tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg
     5881 tgaacactat cccatatcac cagctcaccg tctttcattg ccatacgaaa ttccggatga
     5941 gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc
     6001 tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga
     6061 gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg
     6121 gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac
     6181 tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg
     6241 tgcccgatca agcgctagcg gagtgtatac tggcttacta tgttggcact gatgagggtg
     6301 tcagtgaagt gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat
     6361 gt
//
