LOCUS       62056_2385        7774 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7774)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     CDS             1431..1451
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             1461..4358
                     /codon_start=1
                     /label=RfxCas13d
                     /note="RNA-targeting Type VI CRISPR protein from
                     Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
                     2018)"
                     /translation="IEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDARLEKIVEGDSI
                     RSVNEGEAFSAEMADKNAGYKIGNAKFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEK
                     RYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDKDIIGFGKFSTVY
                     TYDEFKDPEHHRAAFNNNDKLINAIKAQYDEFDNFLDNPRLGYFGQAFFSKEGRNYIIN
                     YGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNE
                     LTNSFSKNSAANVNYIAETLGINPAEFAEQYFRFSIMKEQKNLGFNITKLREVMLDRKD
                     MSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEDAKVAAANKSLPDNEKSLSEKDIFVI
                     NLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAG
                     RDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFF
                     KDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTNLSYDELKALADTFSLDEN
                     GNKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQK
                     KQGQNGKNQIDRYYETCIGKDKGKSVSEKVDALTKIITGMNYDQFDKKRSVIEDTGREN
                     AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEE
                     KGFSSVTKLCAGIDETAPDKRKDVEKEMAERAKESIDSLESANPKLYANYIKYSDEKKA
                     EEFTRQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHA
                     YINDIAEVNSYFQLYHYIMQRIIMNERYEKSSGKVSEYFDAVNDEKKYNDRLLKLLCVP
                     FGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS"
     CDS             4368..4394
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     CDS             4395..4415
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     polyA_signal    4431..4565
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        4572..4812
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        4822..4851
                     /label=RfxCas13d DR30
                     /note="5' processed 30-base direct repeat for an RfxCas13d
                     gRNA (Konermann et al., 2018)"
     misc_RNA        4882..4917
                     /label=RfxCas13d DR36
                     /note="36-base direct repeat for an RfxCas13d pre-gRNA 
                     (Konermann et al., 2018)"
     repeat_region   5037..5177
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5252..5707
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        5989..6093
                     /label=AmpR promoter
     CDS             6094..6951
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7125..7713
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtacgaccgg tgccaccatg cctaaaaaga
     1441 aaagaaaggt gggttctggt atcgaaaaaa aaaagtcctt cgccaagggc atgggcgtga
     1501 agtccacact cgtgtccggc tccaaagtgt acatgacaac cttcgccgaa ggcagcgacg
     1561 ccaggctgga aaagatcgtg gagggcgaca gcatcaggag cgtgaatgag ggcgaggcct
     1621 tcagcgctga aatggccgat aaaaacgccg gctataagat cggcaacgcc aaattcagcc
     1681 atcctaaggg ctacgccgtg gtggctaaca accctctgta tacaggaccc gtccagcagg
     1741 atatgctcgg cctgaaggaa actctggaaa agaggtactt cggcgagagc gctgatggca
     1801 atgacaatat ttgtatccag gtgatccata acatcctgga cattgaaaaa atcctcgccg
     1861 aatacattac caacgccgcc tacgccgtca acaatatctc cggcctggat aaggacatta
     1921 ttggattcgg caagttctcc acagtgtata cctacgacga attcaaagac cccgagcacc
     1981 atagggccgc tttcaacaat aacgataagc tcatcaacgc catcaaggcc cagtatgacg
     2041 agttcgacaa cttcctcgat aaccccagac tcggctattt cggccaggcc tttttcagca
     2101 aggagggcag aaattacatc atcaattacg gcaacgaatg ctatgacatt ctggccctcc
     2161 tgagcggact gaggcactgg gtggtccata acaacgaaga agagtccagg atctccagga
     2221 cctggctcta caacctcgat aagaacctcg acaacgaata catctccacc ctcaactacc
     2281 tctacgacag gatcaccaat gagctgacca actccttctc caagaactcc gccgccaacg
     2341 tgaactatat tgccgaaact ctgggaatca accctgccga attcgccgaa caatatttca
     2401 gattcagcat tatgaaagag cagaaaaacc tcggattcaa tatcaccaag ctcagggaag
     2461 tgatgctgga caggaaggat atgtccgaga tcaggaaaaa tcataaggtg ttcgactcca
     2521 tcaggaccaa ggtctacacc atgatggact ttgtgattta taggtattac atcgaagagg
     2581 atgccaaggt ggctgccgcc aataagtccc tccccgataa tgagaagtcc ctgagcgaga
     2641 aggatatctt tgtgattaac ctgaggggct ccttcaacga cgaccagaag gatgccctct
     2701 actacgatga agctaataga atttggagaa agctcgaaaa tatcatgcac aacatcaagg
     2761 aatttagggg aaacaagaca agagagtata agaagaagga cgcccctaga ctgcccagaa
     2821 tcctgcccgc tggccgtgat gtttccgcct tcagcaaact catgtatgcc ctgaccatgt
     2881 tcctggatgg caaggagatc aacgacctcc tgaccaccct gattaataaa ttcgataaca
     2941 tccagagctt cctgaaggtg atgcctctca tcggagtcaa cgctaagttc gtggaggaat
     3001 acgccttttt caaagactcc gccaagatcg ccgatgagct gaggctgatc aagtccttcg
     3061 ctagaatggg agaacctatt gccgatgcca ggagggccat gtatatcgac gccatccgta
     3121 ttttaggaac caacctgtcc tatgatgagc tcaaggccct cgccgacacc ttttccctgg
     3181 acgagaacgg aaacaagctc aagaaaggca agcacggcat gagaaatttc attattaata
     3241 acgtgatcag caataaaagg ttccactacc tgatcagata cggtgatcct gcccacctcc
     3301 atgagatcgc caaaaacgag gccgtggtga agttcgtgct cggcaggatc gctgacatcc
     3361 agaaaaaaca gggccagaac ggcaagaacc agatcgacag gtactacgaa acttgtatcg
     3421 gaaaggataa gggcaagagc gtgagcgaaa aggtggacgc tctcacaaag atcatcaccg
     3481 gaatgaacta cgaccaattc gacaagaaaa ggagcgtcat tgaggacacc ggcagggaaa
     3541 acgccgagag ggagaagttt aaaaagatca tcagcctgta cctcaccgtg atctaccaca
     3601 tcctcaagaa tattgtcaat atcaacgcca ggtacgtcat cggattccat tgcgtcgagc
     3661 gtgatgctca actgtacaag gagaaaggct acgacatcaa tctcaagaaa ctggaagaga
     3721 agggattcag ctccgtcacc aagctctgcg ctggcattga tgaaactgcc cccgataaga
     3781 gaaaggacgt ggaaaaggag atggctgaaa gagccaagga gagcattgac agcctcgaga
     3841 gcgccaaccc caagctgtat gccaattaca tcaaatacag cgacgagaag aaagccgagg
     3901 agttcaccag gcagattaac agggagaagg ccaaaaccgc cctgaacgcc tacctgagga
     3961 acaccaagtg gaatgtgatc atcagggagg acctcctgag aattgacaac aagacatgta
     4021 ccctgttcag aaacaaggcc gtccacctgg aagtggccag gtatgtccac gcctatatca
     4081 acgacattgc cgaggtcaat tcctacttcc aactgtacca ttacatcatg cagagaatta
     4141 tcatgaatga gaggtacgag aaaagcagcg gaaaggtgtc cgagtacttc gacgctgtga
     4201 atgacgagaa gaagtacaac gataggctcc tgaaactgct gtgtgtgcct ttcggctact
     4261 gtatccccag gtttaagaac ctgagcatcg aggccctgtt cgataggaac gaggccgcca
     4321 agttcgacaa ggagaaaaag aaggtgtccg gcaattccgg ttctggttac ccatacgacg
     4381 taccagatta cgctcccaag aagaagcgca aggtgggata aagatcctaa aacttgttta
     4441 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4501 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     4561 ggatcgcggc cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     4621 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     4681 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     4741 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     4801 tgtggaaagg acgaaacacc gaacccctac caactggtcg gggtttgaaa cggggaagct
     4861 acactgttgt ccaagccatc acaagtaaac ccctaccaac tggtcggggt ttgaaacaca
     4921 ggagctggag atgccccctg aggcacccaa gtaaacccct accaactggt cggggtttga
     4981 aacttttttt gaattctgat gcggtggcgg ccgccacgtg cggaccgagc ggccgcagga
     5041 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     5101 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     5161 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     5221 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     5281 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     5341 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5401 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5461 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5521 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5581 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     5641 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     5701 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     5761 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     5821 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     5881 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     5941 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     6001 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     6061 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6121 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6181 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6241 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6301 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6361 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6421 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6481 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6541 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6601 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6661 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6721 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6781 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6841 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6901 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6961 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     7021 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     7081 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7141 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7201 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7261 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7321 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7381 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7441 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7501 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7561 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7621 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7681 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7741 ttcctggcct tttgctggcc ttttgctcac atgt
//
