LOCUS       Exported                7722 bp ds-DNA     circular SYN 28-JUL-2022
DEFINITION  pJUL2497.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-PE2.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7722)
  TITLE     Liu Lab plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7722)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Jul 29, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7722
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /note="color: #ffffff"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="/Color=#99ff99"
                     /note="/modified_by=jsfg"
                     /note="color: #a6acb3"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /note="color: #ffffff"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff"
     regulatory      649..660
                     /label=KOZAK
                     /note="/created_by=jsfg"
                     /note="color: #ffffff"
     misc_feature    658..714
                     /label=bpNLS
                     /label=nonstandard type:  NLS
                     /note="/created_by=jsfg"
                     /note="color: #a6acb3"
     CDS             715..4815
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from 
                     theStreptococcus pyogenes Type II CRISPR/Cas system"
                     /label=SpCas9 (H840A)
                     /note="generates RNA-guided single strand nicks in DNA
                     dueto the H840A mutation in the HNH catalytic domain"
                     /note="/Color=#00cc55"
                     /note="/modified_by=jsfg"
                     /note="color: #993366"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     variation       3229..3231
                     /label=H840A
                     /label=nonstandard type:  mutation
                     /note="/created_by=jsfg"
                     /note="color: #a6acb3"
     misc_feature    4816..4914
                     /label=33AA linker = XTEN with flanking GlySers
                     /label=nonstandard type:  linker
                     /note="/created_by=jsfg"
                     /note="/modified_by=jsfg"
                     /note="color: #a6acb3"
     misc_feature    4915..4926
                     /label=linker
                     /label=nonstandard type:  linker
                     /note="/created_by=jsfg"
                     /note="/modified_by=jsfg"
                     /note="color: #a6acb3"
     misc_feature    4927..4977
                     /label=bpNLS
                     /label=nonstandard type:  NLS
                     /note="/created_by=jsfg"
                     /note="color: #a6acb3"
     regulatory      4978..4980
                     /label=STOP
                     /note="/Color=#000000"
                     /note="/created_by=jsfg"
                     /note="color: #ffffff"
     CDS             4986..5003
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #993366"
                     /translation="HHHHHH"
     primer_bind     complement(5026..5043)
                     /label=BGH-rev
                     /note="color: black; sequence: TAGAAGGCACAGTCGAGG"
     polyA_signal    5032..5256
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     protein_bind    5351..5367
                     /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(5375..5405)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="color: #ffffff; direction: LEFT"
     protein_bind    5420..5441
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the
                     presenceof cAMP."
                     /note="color: #31849b"
     rep_origin      complement(5729..6317)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="/Color=#99ffff"
                     /note="/modified_by=jsfg"
                     /note="color: #ffff00"
     CDS             complement(6488..7348)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /note="/Color=#ffdd55"
                     /note="/modified_by=jsfg"
                     /note="color: #993366"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7111..7130
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
                     /note="color: #a020f0; direction: RIGHT"
     promoter        complement(7349..7453)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      601 agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc cgccaccatg
      661 aaacggacag ccgacggaag cgagttcgag tcaccaaaga agaagcggaa agtcgacaag
      721 aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac
      781 gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc
      841 aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg
      901 ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa
      961 gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag
     1021 tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg
     1081 gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg
     1141 gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag
     1201 ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag
     1261 ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc
     1321 agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa
     1381 aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc
     1441 ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa
     1501 ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc
     1561 gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc
     1621 gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag
     1681 agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg
     1741 cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt
     1801 gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg
     1861 gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg
     1921 accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg
     1981 cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc
     2041 ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc
     2101 tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac
     2161 aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc
     2221 aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag
     2281 ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag
     2341 cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag
     2401 ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg
     2461 gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac
     2521 aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg
     2581 acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc
     2641 gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc
     2701 cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg
     2761 aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc
     2821 tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac
     2881 attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg
     2941 gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg
     3001 gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg
     3061 atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac
     3121 acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac
     3181 gtggaccagg aactggacat caaccggctg tccgactacg atgtggacgc tatcgtgcct
     3241 cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac
     3301 cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg
     3361 cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc
     3421 gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa
     3481 acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac
     3541 gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg
     3601 tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac
     3661 gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag
     3721 ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc
     3781 aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg
     3841 aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc
     3901 gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg
     3961 cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc
     4021 ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag
     4081 aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg
     4141 ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg
     4201 ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa
     4261 gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg
     4321 ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga
     4381 aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag
     4441 aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag
     4501 cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc
     4561 gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga
     4621 gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc
     4681 ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg
     4741 gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct
     4801 cagctgggag gtgactctgg aggatctagc ggaggatcct ctggcagcga gacaccagga
     4861 acaagcgagt cagcaacacc agagagcagt ggcggcagca gcggcggcag cagctctggc
     4921 ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtctaa
     4981 ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt
     5041 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     5101 ccactcccac tgtcctttcc taataaaatg agaaaattgc atcgcattgt ctgagtaggt
     5161 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     5221 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     5281 ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5341 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5401 gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5461 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5521 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5581 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5641 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5701 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5761 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5821 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5881 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5941 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6001 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6061 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6121 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6181 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6241 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6301 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac actcagtgga
     6361 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6421 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6481 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6541 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6601 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6661 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6721 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6781 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6841 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6901 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6961 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     7021 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     7081 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     7141 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     7201 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7261 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7321 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7381 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7441 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7501 gatctcccga tcccctaggg tctactctca gtacaatctg ctctgatgcc gcatagttaa
     7561 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     7621 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     7681 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg tt
//
