LOCUS       sequence-455012-        6324 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CasRx (RfxCas13d) with double processed direct repeats 
            (15nt) and a non-target spacer in between at 3' end.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6324)
  TITLE     CTRL Enables Gene-specific RNA Regulation Using CRISPR-Cas7-11
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6324)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: 3'dmDR-CMV-CasRx-bGHpolyA
FEATURES             Location/Qualifiers
     source          1..6324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    1062..1286
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1275..1292
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1315..1332)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1394..4294)
                     /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"
     promoter        complement(4336..4539)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4365..4385)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4540..4919
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5091..5110
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5189..5293
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5294..5362,5363..6154)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5512..5531)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctcttcccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcatcctag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct agctagaggt
     1021 cgacggtatc gagccccagc tggttctttc cgcctcagaa gccatagagc ccaccgcatc
     1081 cccagcatgc ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc ccaccccacc
     1141 ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat tttattagga
     1201 aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg ggcaaacaac
     1261 agatggctgg caactagaag gcacagtcga ggctgatcag cgggtttaaa ctcaatggtg
     1321 atggtgatga tggttccgtg acatcaaaaa tacaagggct accaggtagg ttccgtgaca
     1381 tcaaaccggt ttaggaattg ccggacacct tctttttctc cttgtcgaac ttggcggcct
     1441 cgttcctatc gaacagggcc tcgatgctca ggttcttaaa cctggggata cagtagccga
     1501 aaggcacaca cagcagtttc aggagcctat cgttgtactt cttctcgtca ttcacagcgt
     1561 cgaagtactc ggacaccttt ccgctgcttt tctcgtacct ctcattcatg ataattctct
     1621 gcatgatgta atggtacagt tggaagtagg aattgacctc ggcaatgtcg ttgatatagg
     1681 cgtggacata cctggccact tccaggtgga cggccttgtt tctgaacagg gtacatgtct
     1741 tgttgtcaat tctcaggagg tcctccctga tgatcacatt ccacttggtg ttcctcaggt
     1801 aggcgttcag ggcggttttg gccttctccc tgttaatctg cctggtgaac tcctcggctt
     1861 tcttctcgtc gctgtatttg atgtaattgg catacagctt ggggttggcg ctctcgaggc
     1921 tgtcaatgct ctccttggct ctttcagcca tctccttttc cacgtccttt ctcttatcgg
     1981 gggcagtttc atcaatgcca gcgcagagct tggtgacgga gctgaatccc ttctcttcca
     2041 gtttcttgag attgatgtcg tagcctttct ccttgtacag ttgagcatca cgctcgacgc
     2101 aatggaatcc gatgacgtac ctggcgttga tattgacaat attcttgagg atgtggtaga
     2161 tcacggtgag gtacaggctg atgatctttt taaacttctc cctctcggcg ttttccctgc
     2221 cggtgtcctc aatgacgctc cttttcttgt cgaattggtc gtagttcatt ccggtgatga
     2281 tctttgtgag agcgtccacc ttttcgctca cgctcttgcc cttatccttt ccgatacaag
     2341 tttcgtagta cctgtcgatc tggttcttgc cgttctggcc ctgttttttc tggatgtcag
     2401 cgatcctgcc gagcacgaac ttcaccacgg cctcgttttt ggcgatctca tggaggtggg
     2461 caggatcacc gtatctgatc aggtagtgga accttttatt gctgatcacg ttattaataa
     2521 tgaaatttct catgccgtgc ttgcctttct tgagcttgtt tccgttctcg tccagggaaa
     2581 aggtgtcggc gagggccttg agctcatcat aggacaggtt ggttcctaaa atacggatgg
     2641 cgtcgatata catggccctc ctggcatcgg caataggttc tcccattcta gcgaaggact
     2701 tgatcagcct cagctcatcg gcgatcttgg cggagtcttt gaaaaaggcg tattcctcca
     2761 cgaacttagc gttgactccg atgagaggca tcaccttcag gaagctctgg atgttatcga
     2821 atttattaat cagggtggtc aggaggtcgt tgatctcctt gccatccagg aacatggtca
     2881 gggcatacat gagtttgctg aaggcggaaa catcacggcc agcgggcagg attctgggca
     2941 gtctaggggc gtccttcttc ttatactctc ttgtcttgtt tcccctaaat tccttgatgt
     3001 tgtgcatgat attttcgagc tttctccaaa ttctattagc ttcatcgtag tagagggcat
     3061 ccttctggtc gtcgttgaag gagcccctca ggttaatcac aaagatatcc ttctcgctca
     3121 gggacttctc attatcgggg agggacttat tggcggcagc caccttggca tcctcttcga
     3181 tgtaatacct ataaatcaca aagtccatca tggtgtagac cttggtcctg atggagtcga
     3241 acaccttatg atttttcctg atctcggaca tatccttcct gtccagcatc acttccctga
     3301 gcttggtgat attgaatccg aggtttttct gctctttcat aatgctgaat ctgaaatatt
     3361 gttcggcgaa ttcggcaggg ttgattccca gagtttcggc aatatagttc acgttggcgg
     3421 cggagttctt ggagaaggag ttggtcagct cattggtgat cctgtcgtag aggtagttga
     3481 gggtggagat gtattcgttg tcgaggttct tatcgaggtt gtagagccag gtcctggaga
     3541 tcctggactc ttcttcgttg ttatggacca cccagtgcct cagtccgctc aggagggcca
     3601 gaatgtcata gcattcgttg ccgtaattga tgatgtaatt tctgccctcc ttgctgaaaa
     3661 aggcctggcc gaaatagccg agtctggggt tatcgaggaa gttgtcgaac tcgtcatact
     3721 gggccttgat ggcgttgatg agcttatcgt tattgttgaa agcggcccta tggtgctcgg
     3781 ggtctttgaa ttcgtcgtag gtatacactg tggagaactt gccgaatcca ataatgtcct
     3841 tatccaggcc ggagatattg ttgacggcgt aggcggcgtt ggtaatgtat tcggcgagga
     3901 ttttttcaat gtccaggatg ttatggatca cctggataca aatattgtca ttgccatcag
     3961 cgctctcgcc gaagtacctc ttttccagag tttccttcag gccgagcata tcctgctgga
     4021 cgggtcctgt atacagaggg ttgttagcca ccacggcgta gcccttagga tggctgaatt
     4081 tggcgttgcc gatcttatag ccggcgtttt tatcggccat ttcagcgctg aaggcctcgc
     4141 cctcattcac gctcctgatg ctgtcgccct ccacgatctt ttccagcctg gcgtcgctgc
     4201 cttcggcgaa ggttgtcatg tacactttgg agccggacac gagtgtggac ttcacgccca
     4261 tgcccttggc gaaggacttt tttttttcga tcatgcggcc gcggatctct agcggatctg
     4321 acggttcact aaaccagctc tgcttatata gacctcccac cgtacacgcc taccgcccat
     4381 ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag tcccgttgat tttggtgcca
     4441 aaacaaactc ccattgacgt caatggggtg gagacttgga aatccccgtg agtcaaaccg
     4501 ctatccacgc ccattgatgt actgccaaaa ccgcatcacc atggtaatag cgatgactaa
     4561 tacgtagatg tactgccaag taggaaagtc ccataaggtc atgtactggg cataatgcca
     4621 ggcgggccat ttaccgtcat tgacgtcaat agggggcgta cttggcatat gatacacttg
     4681 atgtactgcc aagtgggcag tttaccgtaa atactccacc cattgacgtc aatggaaagt
     4741 ccctattggc gttactatgg gaacatacgt cattattgac gtcaatgggc gggggtcgtt
     4801 gggcggtcag ccaggcgggc catttaccgt aagttatgta acgcggaact ccatatatgg
     4861 gctatgaact aatgaccccg taattgatta ctattaataa ctagtcaata atcaatgtca
     4921 acgcgtatat ctggcccgta catcgcgaag cagcgcaaaa cgcctaaccc taagcagatt
     4981 cttcatgcaa ttgtcggtca agccttgcct tgttgtagct taaattttgc tcgcgcacta
     5041 ctcagcgacc tccaacacac aagcagggag cagatactgg cttaactatg cggcatcaga
     5101 gcagattgta ctgagagtcg accctagggg atcgggagat cgatctcccg atccgtcgac
     5161 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5221 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5281 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5341 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5401 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5461 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5521 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5581 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5641 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5701 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5761 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5821 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5881 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     5941 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6001 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6061 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6121 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6181 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6241 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag tgtcagaccc
     6301 cgtagaaaag atcaaaggat cttc
//
