LOCUS       pT3TS-RfxCas13d-        6169 bp DNA     circular SYN 12-FEB-2025
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6169)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 14, 2025 from SnapGene Viewer 7.2.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6169
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     5'UTR           44..86
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
                     /note="color: #a6acb3; direction: RIGHT"
     regulatory      102..111
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
                     /regulatory_class="other"
     CDS             111..140
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     CDS             141..152
                     /label=Flexible linker
                     /note="color: #c0c0c0; direction: RIGHT"
     CDS             153..179
                     /label=oNLS
                     /note="color: #993366; direction: RIGHT"
     CDS             195..3092
                     /codon_start=1
                     /product="RNA-targeting Type VI CRISPR protein from 
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /label=RfxCas13d
                     /note="color: #993366"
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             3096..3122
                     /codon_start=1
                     /label=oNLS
                     /note="color: #993366"
                     /translation="PPPKRPRLD"
     3'UTR           3147..3272
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
                     /note="color: #a6acb3; direction: RIGHT"
     promoter        complement(3359..3377)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(3384..3400)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     rep_origin      complement(3541..3996)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        4023..4127
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             join(4128..4196,4197..4988)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 4128 .. 4196 / #ccffcc / signal sequence
                        2: 4197 .. 4988 / #ccffcc
                     Cleavage site after base 4196"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5159..5747
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     protein_bind    6035..6056
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     promoter        join(6071..6076,6077..6094,6095..6101)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This forward directional feature has 3 segments:
                        1: 6071 .. 6076 / #ffffff / -35
                        2: 6077 .. 6094 / #ffffff
                        3: 6095 .. 6101 / #ffffff / -10"
     protein_bind    6109..6125
                     /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)."
                     /note="color: #31849b"
     primer_bind     6133..6149
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
ORIGIN
        1 aattaaccct cactaaaggg aacaaaagct ggagctaagc ttgcttgttc tttttgcaga
       61 agctcagaat aaacgctcaa ctttggcaga tctaactcga cgccaccatg gagcagaagc
      121 tgatcagcga ggaggacctg ggaggaagcg gaccacctcc caagaggccc aggctggacg
      181 cgagcgaggc cagcatcgaa aaaaaaaagt ccttcgccaa gggcatgggc gtgaagtcca
      241 cactcgtgtc cggctccaaa gtgtacatga caaccttcgc cgaaggcagc gacgccaggc
      301 tggaaaagat cgtggagggc gacagcatca ggagcgtgaa tgagggcgag gccttcagcg
      361 ctgaaatggc cgataaaaac gccggctata agatcggcaa cgccaaattc agccatccta
      421 agggctacgc cgtggtggct aacaaccctc tgtatacagg acccgtccag caggatatgc
      481 tcggcctgaa ggaaactctg gaaaagaggt acttcggcga gagcgctgat ggcaatgaca
      541 atatttgtat ccaggtgatc cataacatcc tggacattga aaaaatcctc gccgaataca
      601 ttaccaacgc cgcctacgcc gtcaacaata tctccggcct ggataaggac attattggat
      661 tcggcaagtt ctccacagtg tatacctacg acgaattcaa agaccccgag caccataggg
      721 ccgctttcaa caataacgat aagctcatca acgccatcaa ggcccagtat gacgagttcg
      781 acaacttcct cgataacccc agactcggct atttcggcca ggcctttttc agcaaggagg
      841 gcagaaatta catcatcaat tacggcaacg aatgctatga cattctggcc ctcctgagcg
      901 gactgaggca ctgggtggtc cataacaacg aagaagagtc caggatctcc aggacctggc
      961 tctacaacct cgataagaac ctcgacaacg aatacatctc caccctcaac tacctctacg
     1021 acaggatcac caatgagctg accaactcct tctccaagaa ctccgccgcc aacgtgaact
     1081 atattgccga aactctggga atcaaccctg ccgaattcgc cgaacaatat ttcagattca
     1141 gcattatgaa agagcagaaa aacctcggat tcaatatcac caagctcagg gaagtgatgc
     1201 tggacaggaa ggatatgtcc gagatcagga aaaatcataa ggtgttcgac tccatcagga
     1261 ccaaggtcta caccatgatg gactttgtga tttataggta ttacatcgaa gaggatgcca
     1321 aggtggctgc cgccaataag tccctccccg ataatgagaa gtccctgagc gagaaggata
     1381 tctttgtgat taacctgagg ggctccttca acgacgacca gaaggatgcc ctctactacg
     1441 atgaagctaa tagaatttgg agaaagctcg aaaatatcat gcacaacatc aaggaattta
     1501 ggggaaacaa gacaagagag tataagaaga aggacgcccc tagactgccc agaatcctgc
     1561 ccgctggccg tgatgtttcc gccttcagca aactcatgta tgccctgacc atgttcctgg
     1621 atggcaagga gatcaacgac ctcctgacca ccctgattaa taaattcgat aacatccaga
     1681 gcttcctgaa ggtgatgcct ctcatcggag tcaacgctaa gttcgtggag gaatacgcct
     1741 ttttcaaaga ctccgccaag atcgccgatg agctgaggct gatcaagtcc ttcgctagaa
     1801 tgggagaacc tattgccgat gccaggaggg ccatgtatat cgacgccatc cgtattttag
     1861 gaaccaacct gtcctatgat gagctcaagg ccctcgccga caccttttcc ctggacgaga
     1921 acggaaacaa gctcaagaaa ggcaagcacg gcatgagaaa tttcattatt aataacgtga
     1981 tcagcaataa aaggttccac tacctgatca gatacggtga tcctgcccac ctccatgaga
     2041 tcgccaaaaa cgaggccgtg gtgaagttcg tgctcggcag gatcgctgac atccagaaaa
     2101 aacagggcca gaacggcaag aaccagatcg acaggtacta cgaaacttgt atcggaaagg
     2161 ataagggcaa gagcgtgagc gaaaaggtgg acgctctcac aaagatcatc accggaatga
     2221 actacgacca attcgacaag aaaaggagcg tcattgagga caccggcagg gaaaacgccg
     2281 agagggagaa gtttaaaaag atcatcagcc tgtacctcac cgtgatctac cacatcctca
     2341 agaatattgt caatatcaac gccaggtacg tcatcggatt ccattgcgtc gagcgtgatg
     2401 ctcaactgta caaggagaaa ggctacgaca tcaatctcaa gaaactggaa gagaagggat
     2461 tcagctccgt caccaagctc tgcgctggca ttgatgaaac tgcccccgat aagagaaagg
     2521 acgtggaaaa ggagatggct gaaagagcca aggagagcat tgacagcctc gagagcgcca
     2581 accccaagct gtatgccaat tacatcaaat acagcgacga gaagaaagcc gaggagttca
     2641 ccaggcagat taacagggag aaggccaaaa ccgccctgaa cgcctacctg aggaacacca
     2701 agtggaatgt gatcatcagg gaggacctcc tgagaattga caacaagaca tgtaccctgt
     2761 tcagaaacaa ggccgtccac ctggaagtgg ccaggtatgt ccacgcctat atcaacgaca
     2821 ttgccgaggt caattcctac ttccaactgt accattacat catgcagaga attatcatga
     2881 atgagaggta cgagaaaagc agcggaaagg tgtccgagta cttcgacgct gtgaatgacg
     2941 agaagaagta caacgatagg ctcctgaaac tgctgtgtgt gcctttcggc tactgtatcc
     3001 ccaggtttaa gaacctgagc atcgaggccc tgttcgatag gaacgaggcc gccaagttcg
     3061 acaaggagaa aaagaaggtg tccggcaatt ccggacctcc tcccaagagg cccaggctgg
     3121 actgaccgcg gatctggtta ccactaaacc agcctcaaga acacccgaat ggagtctcta
     3181 agctacataa taccaactta cactttacaa aatgttgtcc cccaaaatgt agccattcgt
     3241 atctgctcct aataaaaaga aagtttcttc acattctaaa aaaaaaaaaa aaaaaaaaaa
     3301 aaaaaacccc cccccccccc tgcagggcca agtcggcctc tagaggtacc caattcgccc
     3361 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     3421 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     3481 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     3541 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     3601 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     3661 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     3721 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     3781 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     3841 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     3901 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     3961 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg
     4021 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4081 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4141 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4201 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4261 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4321 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4381 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4441 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4501 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4561 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4621 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     4681 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4741 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4801 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     4861 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     4921 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     4981 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5041 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5101 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5161 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5221 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5281 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     5341 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5401 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5461 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5521 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5581 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5641 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5701 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5761 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     5821 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     5881 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     5941 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     6001 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     6061 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     6121 aacaatttca cacaggaaac agctatgacc atgattacgc caagctcga
//
