LOCUS       Exported                7149 bp ds-DNA     circular SYN 21-SEP-2021
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 7149)
  AUTHORS   Welker Lab
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Sep 21, 2021 from SnapGene 4.3.7
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7149
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     promoter        82..293
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     regulatory      313..317
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
     CDS             355..375
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /note="color: #993366"
                     /translation="PKKKRKV"
     CDS             376..873
                     /codon_start=1
                     /label=tadA ABEe
                     /note="color: #993366"
                     /translation="SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWN
                     RAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFG
                     VRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQS
                     SIN"
     CDS             898..942
                     /codon_start=1
                     /label=XTEN
                     /note="color: #993366"
                     /translation="SGSETPGTSESATPE"
     CDS             970..5070
                     /codon_start=1
                     /label=nHeF-SpCas9
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /note="This feature has 17 segments:
                         1:  970 ..  993 / #993366
                         2:  994 ..  996 / #ff99cc / D10A
                         3:  997 .. 2454 / #993366
                         4: 2455 .. 2457 / #ff99cc / N497A
                         5: 2458 .. 2946 / #993366
                         6: 2947 .. 2949 / #ff99cc / R661A
                         7: 2950 .. 3048 / #993366
                         8: 3049 .. 3051 / #ff99cc / Q695A
                         9: 3052 .. 3507 / #993366
                        10: 3508 .. 3510 / #cc99b2 / K848A
                        11: 3511 .. 3741 / #993366
                        12: 3742 .. 3744 / #ff99cc / Q926A
                        13: 3745 .. 3972 / #993366
                        14: 3973 .. 3975 / #cc99b2 / K1003A
                        15: 3976 .. 4143 / #993366
                        16: 4144 .. 4146 / #cc99b2 / R1060A
                        17: 4147 .. 5070 / #993366"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5071..5118
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /note="color: #993366"
                     /translation="KRPAATKKAGQAKKKK"
     polyA_signal    5152..5359
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     rep_origin      complement(5424..6012)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(6183..7043)
                     /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: 6183 .. 6974 / #ccffcc
                        2: 6975 .. 7043 / #ccffcc / signal sequence
                     Cleavage site after base 6974"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7044..7148)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
ORIGIN
        1 acatttcccc gaaaagtgcc acctgacgtc cagcagagat ccactttggc gccggctcga
       61 gtggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
      121 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
      181 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
      241 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtgtcgt
      301 gacgcgggat ccgccaccat gaaacggaca gccgacggaa gcgagttcga gtcaccaaag
      361 aagaagcgga aagtctctga ggtggagttt tcccacgagt actggatgag acatgccctg
      421 accctggcca agagggcacg ggatgagagg gaggtgcctg tgggagccgt gctggtgctg
      481 aacaatagag tgatcggcga gggctggaac agagccatcg gcctgcacga cccaacagcc
      541 catgccgaaa ttatggccct gagacagggc ggcctggtca tgcagaacta cagactgatt
      601 gacgccaccc tgtacgtgac attcgagcct tgcgtgatgt gcgccggcgc catgatccac
      661 tctaggatcg gccgcgtggt gtttggcgtg aggaactcaa aaagaggcgc cgcaggctcc
      721 ctgatgaacg tgctgaacta ccccggcatg aatcaccgcg tcgaaattac cgagggaatc
      781 ctggcagatg aatgtgccgc cctgctgtgc gatttctatc ggatgcctag acaggtgttc
      841 aatgctcaga agaaggccca gagctccatc aactccggag gatctagcgg aggctcctct
      901 ggctctgaga cacctggcac aagcgagagc gcaacacctg aatcctcagg tggtagctct
      961 ggggggtctg acaagaagta cagcatcggc ctggccatcg gcaccaactc tgtgggctgg
     1021 gccgtgatca ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc
     1081 gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca
     1141 gccgaggcca cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg
     1201 atctgctatc tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc
     1261 cacagactgg aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc
     1321 ttcggcaaca tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg
     1381 agaaagaaac tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg
     1441 gcccacatga tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac
     1501 agcgacgtgg acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa
     1561 aaccccatca acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag
     1621 agcagacggc tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc
     1681 ggaaacctga ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg
     1741 gccgaggatg ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg
     1801 ctggcccaga tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac
     1861 gccatcctgc tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc
     1921 gcctctatga tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc
     1981 gtgcggcagc agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc
     2041 tacgccggct acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc
     2101 atcctggaaa agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg
     2161 ctgcggaagc agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag
     2221 ctgcacgcca ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa
     2281 aagatcgaga agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga
     2341 aacagcagat tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc
     2401 gaggaagtgg tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccgccttc
     2461 gataagaacc tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc
     2521 accgtgtata acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc
     2581 ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa
     2641 gtgaccgtga agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg
     2701 gaaatctccg gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg
     2761 aaaattatca aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat
     2821 atcgtgctga ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc
     2881 tatgcccacc tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc
     2941 tggggcgcgc tgagccggaa gctgatcaac ggcatccgcg acaagcagtc cggcaagaca
     3001 atcctggatt tcctgaagtc cgacggcttc gccaacagaa acttcatggc gctgatccac
     3061 gacgacagcc tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat
     3121 agcctgcacg agcacattgc caatctcgcc ggcagccccg ccattaagaa gggcatcctc
     3181 cagacagtga aggtggtgga cgagctggtg aaagtgatgg gccggcacaa gcccgagaac
     3241 atcgtgatcg aaatggccag agagaaccag accacccaga agggacagaa gaacagtcgc
     3301 gagagaatga agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa
     3361 caccccgtgg aaaacaccca gctccagaac gagaagctgt acctgtacta cctccagaat
     3421 gggcgggata tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg
     3481 gaccatatcg tgcctcagag ctttctggcg gacgactcca tcgacaacaa ggtgctgacc
     3541 agaagcgaca agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag
     3601 atgaagaact actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac
     3661 aatctgacca aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag
     3721 agacagctgg tggaaacccg ggcgatcaca aagcacgtgg cacagatcct ggactcccgg
     3781 atgaacacta agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg
     3841 aagtccaagc tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc
     3901 aacaactacc accacgccca cgacgcgtac ctgaacgccg tcgtgggaac cgccctgatc
     3961 aaaaagtacc ctgcgctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgta
     4021 cggaagatga tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc
     4081 tacagcaaca tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg
     4141 aaggcgcctc tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg
     4201 gattttgcca ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc
     4261 gaggtgcaga caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag
     4321 ctgatcgcca gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc
     4381 gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag
     4441 agtgtgaaag agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc
     4501 atcgactttc tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg
     4561 cctaagtact ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc
     4621 gaactgcaga agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg
     4681 gccagccact atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt
     4741 gtggaacagc acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag
     4801 agagtgatcc tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg
     4861 gataagccca tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg
     4921 ggagcccctg ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc
     4981 accaaagagg tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca
     5041 cggatcgacc tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc
     5101 caggcaaaaa agaaaaagta agaattccta gagctcgctg atcagcctcg actgtgcctt
     5161 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     5221 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     5281 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga
     5341 atagcaggca tgctggggag cggccgctga gcaaaaggcc agcaaaaggc caggaaccgt
     5401 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5461 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5521 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5581 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5641 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5701 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5761 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5821 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5881 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5941 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6001 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6061 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6121 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6181 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6241 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6301 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6361 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6421 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6481 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6541 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6601 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6661 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6721 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6781 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6841 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6901 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6961 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7021 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7081 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7141 gttccgcgc
//
