LOCUS       eTd-CBE        8778 bp DNA     circular SYN 07-NOV-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8778)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 7, 2022 from SnapGene 6.1.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8778
                     /mol_type="other DNA"
                     /note="color: #ffffff"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     CDS             964..5064
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due
                     to the D10A mutation in the RuvC catalytic domain"
                     /note="This feature has 4 segments:
                        1:  964 ..  987 / #993366
                        2:  988 ..  990 / #cc99b2
                        3:  991 .. 4601 / #993366
                        4: 4602 .. 5064 / #993366"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5793..5810
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     polyA_signal    5839..6063
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(6134..6150)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    6158..6174
                     /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"
     promoter        complement(6182..6212)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 6182 .. 6188 / #ffffff / -10
                        2: 6189 .. 6206 / #ffffff
                        3: 6207 .. 6212 / #ffffff / -35"
     protein_bind    6227..6248
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /note="color: #31849b"
     rep_origin      complement(6536..7124)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(7295..8155)
                     /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: 7295 .. 8086 / #ccffcc
                        2: 8087 .. 8155 / #ccffcc / signal sequence
                     Cleavage site after base 8086"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(8156..8260)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
     enhancer        join(8530..8778,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #ffffff"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga ggtggagttt
      481 tcccacgagt actggatgag acatgccctg accctggcca agagggcacg ggatgagagg
      541 gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggctg
      601 agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc
      661 ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct
      721 tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg
      781 aggaactcaa aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg
      841 aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc
      901 gatttctatc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc
      961 aacgacaaga agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg
     1021 atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg
     1081 cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag
     1141 gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc
     1201 tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga
     1261 ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
     1321 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag
     1381 aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac
     1441 atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     1501 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc
     1561 atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
     1621 cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac
     1681 ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     1741 gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     1801 cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc
     1861 ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
     1921 atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg
     1981 cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc
     2041 ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg
     2101 gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg
     2161 aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
     2221 gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc
     2281 gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc
     2341 agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa
     2401 gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag
     2461 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     2521 tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg
     2581 agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc
     2641 gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc
     2701 tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt
     2761 atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
     2821 ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc
     2881 cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc
     2941 aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg
     3001 gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac
     3061 agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
     3121 cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca
     3181 gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg
     3241 atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga
     3301 atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
     3361 gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
     3421 gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat
     3481 atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc
     3541 gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag
     3601 aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
     3661 accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
     3721 ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac
     3781 actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc
     3841 aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac
     3901 taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
     3961 taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
     4021 atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc
     4081 aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg
     4141 cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt
     4201 gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
     4261 cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
     4321 gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc
     4381 tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg
     4441 aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac
     4501 tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
     4561 tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
     4621 cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc
     4681 cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa
     4741 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg
     4801 atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
     4861 cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
     4921 cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa
     4981 gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc
     5041 gacctgtctc agctgggagg tgactctggc ggctcaaaaa gaaccgccga cggcagcgaa
     5101 ttcgagccca agaagaagag gaaagtcgga agcggagcta ctaacttcag cctgctgaag
     5161 caggctggag acgtggagga gaaccctgga cctactaatc tgagcgacat cattgagaag
     5221 gagactggga aacagctggt cattcaggag tccatcctga tgctgcctga ggaggtggag
     5281 gaagtgatcg gcaacaagcc agagtctgac atcctggtgc acaccgccta cgacgagtcc
     5341 acagatgaga atgtgatgct gctgacctct gacgcccccg agtataagcc ttgggccctg
     5401 gtcatccagg attctaacgg cgagaataag atcaagatgc tgagcggagg atccggagga
     5461 tctggaggca gcaccaacct gtctgacatc atcgagaagg agacaggcaa gcagctggtc
     5521 atccaggaga gcatcctgat gctgcccgaa gaagtcgaag aagtgatcgg aaacaagcct
     5581 gagagcgata tcctggtcca taccgcctac gacgagagta ccgacgaaaa tgtgatgctg
     5641 ctgacatccg acgccccaga gtataagccc tgggctctgg tcatccagga ttccaacgga
     5701 gagaacaaaa tcaaaatgct gtctggcggc tcaaaaagaa ccgccgacgg cagcgaattc
     5761 gagcccaaga agaagaggaa agtctaaccg gtcatcatca ccatcaccat tgagtttaaa
     5821 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     5881 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     5941 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     6001 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     6061 tggcttctga ggcggaaaga accagctggg gctcgatacc gtcgacctct agctagagct
     6121 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     6181 acaacatacg agccggaagc ataaagtgta aagcctaggg tgcctaatga gtgagctaac
     6241 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6301 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     6361 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6421 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     6481 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6541 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6601 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6661 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6721 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6781 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6841 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6901 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6961 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     7021 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     7081 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     7141 tttctacggg gtctgacact cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     7201 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     7261 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7321 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7381 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7441 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7501 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7561 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7621 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7681 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7741 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7801 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7861 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7921 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7981 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     8041 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     8101 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     8161 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     8221 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     8281 cacctgacgt cgacggatcg ggagatcgat ctcccgatcc cctagggtcg actctcagta
     8341 caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg
     8401 tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt
     8461 gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat
     8521 atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
     8581 ttcatagccc atatattgag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     8641 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     8701 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     8761 cagtacatca agtgtatc
//
