LOCUS       sequence-381538-        6765 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses human codon optimized NcCas9. Genome editing in human 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6765)
  AUTHORS   Liu Z, Chen S, Xie W, Yu H, Lai L, Li Z
  TITLE     Versatile and efficient genome editing with Neisseria cinerea Cas9.
  JOURNAL   Commun Biol. 2022 Nov 26;5(1):1296. doi: 10.1038/s42003-022-04258-z.
  PUBMED    36435853
REFERENCE   2  (bases 1 to 6765)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Commun 
            Biol."; date: "2022-11-26"; volume: "5(1):1296. doi"; pages: " 
            10.1038/s42003-022-04258-z"
COMMENT     Sequence Label: NcCas9
FEATURES             Location/Qualifiers
     source          1..6765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3751..3771
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3780..3797
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3820..3837)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3826..4050
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4121..4137)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4121..4137)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4134..4156)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4145..4161
                     /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)."
     promoter        complement(join(4169..4175,4176..4193,4194..4199))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4214..4235
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4352..4369)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4523..5111)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4603..4622)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5282..6073,6074..6142))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5905..5924
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6143..6247)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6326..6345)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6517..6765,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
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 aagtcgccgc cttcaaacca
      481 aaccctatga actacatcct ggggctggac atcggaatcg caagcgtggg ctgggctatc
      541 gtggaaatcg acgaggagga gaacccaatc cggctgatcg acctgggcgt gagagtgttt
      601 gagagggcag aggtgccaaa gaccggcgat tccctggcag cagcacggag actggcacgg
      661 tctgtgaggc gcctgacacg gagaagggca cacagactgc tgagggcacg ccggctgctg
      721 aagagagagg gcgtgctgca ggcagcagac ttcgatgaga acggcctgat caagagcctg
      781 cctaataccc catggcagct gagagcagcc gccctggaca ggaagctgac accactggag
      841 tggtccgccg tgctgctgca cctgatcaag caccgcggct acctgtctca gcggaagaat
      901 gagggcgaga cagccgataa ggagctgggc gccctgctga agggagtggc cgacaacaca
      961 cacgcactgc agaccggcga cttccggaca cctgcagagc tggccctgaa taagttcgag
     1021 aaggagagcg gacacatcag gaaccagagg ggcgactatt cccacacctt caaccgcaag
     1081 gatctgcagg ccgagctgaa tctgctgttt gagaagcaga aggagttcgg caatcctcac
     1141 gtgtccgacg gcctgaagga gggcatcgag acactgctga tgacccagag gccagccctg
     1201 tctggcgatg cagtgcagaa gatgctgggc cactgcacat ttgagcccac cgagcctaag
     1261 gccgccaaga acacctacac agccgagagg ttcgtgtggc tgacaaagct gaacaatctg
     1321 aggatcctgg agcagggaag cgagaggcct ctgaccgaca cagagagggc caccctgatg
     1381 gatgagccat acaggaagtc caagctgaca tatgcacagg caaggaagct gctggacctg
     1441 gacgataccg ccttctttaa gggcctgaga tacggcaagg ataatgccga ggcctctaca
     1501 ctgatggaga tgaaggccta tcacgccatc agccgcgccc tggagaagga gggcctgaag
     1561 gacaagaagt ctcctctgaa cctgagccca gagctgcagg atgagatcgg caccgccttt
     1621 tccctgttca agaccgacga ggacatcaca ggccgcctga aggacagggt gcagcctgag
     1681 atcctggagg ccctgctgaa gcacatctct tttgataagt tcgtgcagat cagcctgaag
     1741 gccctgagaa ggatcgtgcc actgatggag cagggcaatc gctacgacga ggcctgcacc
     1801 gagatctacg gcgatcacta tggcaagaag aacacagagg agaagatcta tctgccccct
     1861 atcccagccg acgagatcag aaatccagtg gtgctgaggg ccctgagcca ggcacgcaaa
     1921 gtgatcaacg gagtggtgcg ccggtacggc tcccccgcaa ggatccacat cgagacagcc
     1981 agagaagtgg gcaagtcttt taaggaccgg aaggagatcg agaagagaca ggaggagaat
     2041 agaaaggata gggagaagag cgccgccaag ttcagggagt actttcccaa cttcgtgggc
     2101 gagcctaagt ccaaggacat cctgaagctg cgcctgtacg agcagcagca cggcaagtgt
     2161 ctgtatagcg gcaaggagat caacctgggc cggctgaatg agaagggcta tgtggagatc
     2221 gatcacgccc tgccctttag cagaacctgg gacgattcct tcaacaataa ggtgctggcc
     2281 ctgggctctg agaaccagaa taagggcaat cagacaccat acgagtattt caacggcaag
     2341 gacaatagca gggagtggca ggagtttaag gcaagggtgg agacaagcag gttcccacgg
     2401 tctaagaagc agcggatcct gctgcagaag tttgacgagg atggcttcaa ggagagaaac
     2461 ctgaatgaca ccagatacat caacaggttt ctgtgccagt tcgtggccga tcacatgctg
     2521 ctgaccggca agggcaagag aagggtgttt gcctccaacg gccagatcac aaatctgctg
     2581 aggggcttct ggggcctgag aaaggtgagg gcagagaatg acaggcacca cgcactggat
     2641 gcagtggtgg tggcatgtag caccatcgcc atgcagcaga agatcacacg ctttgtgcgg
     2701 tataaggaga tgaacgcctt cgacggcaag accatcgata aggagacagg cgaggtgctg
     2761 caccagaagg cccactttcc acagccctgg gagttctttg cccaggaagt gatgatccgg
     2821 gtgtttggca agccagacgg caagcccgag ttcgaggagg ccgatacccc agagaagctg
     2881 cggacactgc tggcagagaa gctgagctcc agacctgagg ccgtgcacaa gtacgtgacc
     2941 cctctgttca tctcccgcgc cccaaatcgg aagatgtctg gccagggcca catggagaca
     3001 gtgaagtctg ccaagcggct ggacgagggc atcagcgtgc tgagagtgcc cctgacacag
     3061 ctgaagctga aggatctgga gaagatggtg aaccgcgagc gggagcctaa gctgtatgag
     3121 gccctgaagg ccagactgga ggcccacaag gacgatcccg ccaaggcctt tgccgagcct
     3181 ttctacaagt atgacaaggc cggcaatcgg acccagcagg tgaaggccgt gagagtggag
     3241 caggtgcaga agacaggcgt gtgggtgcac aaccacaatg gcatcgccga caacgccaca
     3301 atcgtgcggg tggacgtgtt cgagaagggc ggcaagtact atctggtgcc aatctactcc
     3361 tggcaggtgg caaagggaat cctgccagac agagccgtgg tgcagggcaa ggacgaggag
     3421 gattggaccg tgatggacga ttctttcgag tttaagttcg tgctgtacgc caacgacctg
     3481 atcaagctga ccgccaagaa gaatgagttt ctgggctatt tcgtgtctct gaacagggca
     3541 acaggagcaa tcgacatcag gacccacgac acagatagca ccaagggcaa gaacggcatc
     3601 tttcagagcg tgggcgtgaa gaccgccctg tccttccaga agtatcagat tgacgaactg
     3661 ggaaaagaga ttagaccctg taggctgaaa aagagaccac ctgtgagatc tggcggctca
     3721 aaaagaaccg ccgacggcag cgaattcgag cccaagaaga agaggaaagt ctaaccggtc
     3781 atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     3841 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     3901 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     3961 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     4021 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     4081 cgataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     4141 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     4201 cctagggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     4261 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     4321 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4381 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4441 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4501 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4561 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4621 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4681 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4741 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4801 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4861 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4921 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4981 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5041 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5101 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacactcag tggaacgaaa
     5161 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5221 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5281 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5341 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5401 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5461 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5521 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5581 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5641 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5701 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5761 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5821 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5881 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5941 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6001 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6061 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6121 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6181 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6241 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatcgatctc
     6301 ccgatcccct agggtcgact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     6361 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     6421 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     6481 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
     6541 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     6601 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     6661 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     6721 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatc
//
