LOCUS       sequence-431388-        5501 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (Cas protein) Cas13d.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5501)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 5501)
  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: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: AA076
FEATURES             Location/Qualifiers
     source          1..5501
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             445..3339
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGN"
     primer_bind     complement(3411..3427)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3411..3427)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3424..3446)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3435..3451
                     /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)."
     promoter        complement(join(3459..3465,3466..3483,3484..3489))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3504..3525
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3642..3659)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3813..4401)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3893..3912)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4579..5388)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5302..5321
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5380..5399
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tgaagactgg cagcatcgaa aagaagaagt ccttcgccaa gggcatgggc
      481 gtgaagtcca cactcgtgtc cggctccaaa gtgtacatga caaccttcgc cgaaggcagc
      541 gacgccaggc tggaaaagat cgtggagggc gacagcatca ggagcgtgaa tgagggcgag
      601 gccttcagcg ctgaaatggc cgataaaaac gccggctata agatcggcaa cgccaaattc
      661 agccatccta agggctacgc cgtggtggct aacaaccctc tgtatacagg acccgtccag
      721 caggatatgc tcggcctgaa ggaaactctg gaaaagaggt acttcggcga gagcgctgat
      781 ggcaatgaca atatttgtat ccaggtgatc cataacatcc tggacattga aaaaatcctc
      841 gccgaataca ttaccaacgc cgcctacgcc gtcaacaata tctccggcct ggataaggac
      901 attattggat tcggcaagtt ctccacagtg tatacctacg acgaattcaa agaccccgag
      961 caccataggg ccgctttcaa caataacgat aagctcatca acgccatcaa ggcccagtat
     1021 gacgagttcg acaacttcct cgataacccc agactcggct atttcggcca ggcctttttc
     1081 agcaaggagg gcagaaatta catcatcaat tacggcaacg aatgctatga cattctggcc
     1141 ctcctgagcg gactgaggca ctgggtggtc cataacaacg aagaagagtc caggatctcc
     1201 aggacctggc tctacaacct cgataagaac ctcgacaacg aatacatctc caccctcaac
     1261 tacctctacg acaggatcac caatgagctg accaactcct tctccaagaa ctccgccgcc
     1321 aacgtgaact atattgccga aactctggga atcaaccctg ccgaattcgc cgaacaatat
     1381 ttcagattca gcattatgaa agagcagaaa aacctcggat tcaatatcac caagctcagg
     1441 gaagtgatgc tggacaggaa ggatatgtcc gagatcagga aaaatcataa ggtgttcgac
     1501 tccatcagga ccaaggtcta caccatgatg gactttgtga tttataggta ttacatcgaa
     1561 gaggatgcca aggtggctgc cgccaataag tccctccccg ataatgagaa gtccctgagc
     1621 gagaaggata tctttgtgat taacctgagg ggctccttca acgacgacca gaaggatgcc
     1681 ctctactacg atgaagctaa tagaatttgg agaaagctcg aaaatatcat gcacaacatc
     1741 aaggaattta ggggaaacaa gacaagagag tataagaaga aggacgcccc tagactgccc
     1801 agaatcctgc ccgctggccg tgatgtttcc gccttcagca aactcatgta tgccctgacc
     1861 atgttcctgg atggcaagga gatcaacgac ctcctgacca ccctgattaa taaattcgat
     1921 aacatccaga gcttcctgaa ggtgatgcct ctcatcggag tcaacgctaa gttcgtggag
     1981 gaatacgcct ttttcaaaga ctccgccaag atcgccgatg agctgaggct gatcaagtcc
     2041 ttcgctagaa tgggagaacc tattgccgat gccaggaggg ccatgtatat cgacgccatc
     2101 cgtattttag gaaccaacct gtcctatgat gagctcaagg ccctcgccga caccttttcc
     2161 ctggacgaga acggaaacaa gctcaagaaa ggcaagcacg gcatgagaaa tttcattatt
     2221 aataacgtga tcagcaataa aaggttccac tacctgatca gatacggtga tcctgcccac
     2281 ctccatgaga tcgccaaaaa cgaggccgtg gtgaagttcg tgctcggcag gatcgctgac
     2341 atccagaaaa aacagggcca gaacggcaag aaccagatcg acaggtacta cgaaacttgt
     2401 atcggaaagg ataagggcaa gagcgtgagc gaaaaggtgg acgctctcac aaagatcatc
     2461 accggaatga actacgacca attcgacaag aaaaggagcg tcattgagga caccggcagg
     2521 gaaaacgccg agagggagaa gtttaaaaag atcatcagcc tgtacctcac cgtgatctac
     2581 cacatcctca agaatattgt caatatcaac gccaggtacg tcatcggatt ccattgcgtc
     2641 gagcgtgatg ctcaactgta caaggagaaa ggctacgaca tcaatctcaa gaaactggaa
     2701 gagaagggat tcagctccgt caccaagctc tgcgctggca ttgatgaaac tgcccccgat
     2761 aagagaaagg acgtggaaaa ggagatggct gaaagagcca aggagagcat tgacagcctc
     2821 gagagcgcca accccaagct gtatgccaat tacatcaaat acagcgacga gaagaaagcc
     2881 gaggagttca ccaggcagat taacagggag aaggccaaaa ccgccctgaa cgcctacctg
     2941 aggaacacca agtggaatgt gatcatcagg gaggacctcc tgagaattga caacaagaca
     3001 tgtaccctgt tcagaaacaa ggccgtccac ctggaagtgg ccaggtatgt ccacgcctat
     3061 atcaacgaca ttgccgaggt caattcctac ttccaactgt accattacat catgcagaga
     3121 attatcatga atgagaggta cgagaaaagc agcggaaagg tgtccgagta cttcgacgct
     3181 gtgaatgacg agaagaagta caacgatagg ctcctgaaac tgctgtgtgt gcctttcggc
     3241 tactgtatcc ccaggtttaa gaacctgagc atcgaggccc tgttcgatag gaacgaggcc
     3301 gccaagttcg acaaggagaa aaagaaggtg tccggcaatg ggtccaagtc ttcatcggat
     3361 cccgggcccg tcgactgcag aggcctgcat gcaagcttgg tgtaatcatg gtcatagctg
     3421 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     3481 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     3541 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3601 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3661 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3721 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3781 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     3841 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     3901 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     3961 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4021 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4081 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4141 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4201 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4261 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4321 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4381 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4441 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4501 tagatccttt taaattaaaa atgaagtttt aaatcaagcc caatctgaat aatgttacaa
     4561 ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     4621 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     4681 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     4741 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     4801 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     4861 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     4921 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     4981 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     5041 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gtttttccgg ggatcgcagt
     5101 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     5161 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     5221 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaagc gatagattgt
     5281 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     5341 gttggaattt aatcgcggcc tcgacgtttc ccgttgaata tggctcataa caccccttgt
     5401 attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc
     5461 aatgtaacat cagagatttt gagacacggg ccagagctgc a
//
