LOCUS       sequence-436458-        6305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammlian expression of MmFnuc for genome editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6305)
  AUTHORS   Jiang K, Lim J, Sgrizzi S, Trinh M, Kayabolen A, Yutin N, Bao W, 
            Kato K, Koonin EV, Gootenberg JS, Abudayyeh OO
  TITLE     Programmable RNA-guided DNA endonucleases are widespread in 
            eukaryotes and their viruses.
  JOURNAL   Sci Adv. 2023 Sep 29;9(39):eadk0171. doi: 10.1126/sciadv.adk0171. 
            Epub 2023 Sep 27.
  PUBMED    37756409
REFERENCE   2  (bases 1 to 6305)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2023-09-29"; volume: "9(39):eadk0171. doi"; pages: " 
            10.1126/sciadv.adk0171. Epub 2023 Sep 27"
COMMENT     Sequence Label: pKJ235
FEATURES             Location/Qualifiers
     source          1..6305
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        6..385
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        387..664
                     /label=chicken beta-actin promoter
     intron          665..1681
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     CDS             3149..3196
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(3224..3241)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3230..3454
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3526..3543)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3526..3542)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3535..3557)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(3683..4138)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3820..3841)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4032..4051
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4165..4269
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4270..4338,4339..5130)
                     /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     complement(4488..4507)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5301..5889
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5790..5809
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6043..6060
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6177..6198
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6213..6218,6219..6236,6237..6243)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6251..6267
                     /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)."
     primer_bind     6256..6278
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6275..6291
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6275..6291
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gattcgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
       61 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      121 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      181 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      241 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      301 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      361 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactccccc
      421 catccccccc cccccccccc ccccaatttt gtatttattt attttttaat tattttgggc
      481 agcgatgggg gcgggggggg ggggggggcg cgcgccgggc ggggcggggc ggggcgaggg
      541 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      781 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      841 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
      901 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
      961 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
     1021 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
     1081 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     1141 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     1201 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
     1261 ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc
     1321 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     1381 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     1441 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     1501 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     1561 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1621 tgtgactggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca
     1681 gatccttaat taagccacca tgaaacgaaa gagagaacaa atgacactct ggaaagctgc
     1741 atttgttaac ggacaggaga cgttcaagtc atggattgac aaggcgcgga tgttggaatt
     1801 gaactgcgat gtcagctcag ccagcagtac gcattacagc gatcttaatc tcaagactaa
     1861 gtgcgccaag atggaggata agttcatgtg tacattttca gtaggaatta gaccaacctc
     1921 aaaacaaaag cgcaccctga accagatgct caaggtaagc aatcacgcct ataactggtg
     1981 caactatctc gtgaaagaaa aggacttcaa gccaaaacaa tttgacttgc aacgcgtagt
     2041 gactaaaacg aactcaacag atgtaccagc cgaatatcgg cttcctggcg acgactggtt
     2101 ctttgataac aagatgtcca gcatcaaatt gacagcgtgt aagaatttct gtacgatgta
     2161 caaatctgcg cagaccaacc agaaaaagac gaaggttgat ctcaggaata aagacatagc
     2221 tatgttgcga gaaggtagct tcgaggtcca gaagaaatat gtacgcctcc tgaccgagaa
     2281 agacatccca gacgaacgca tcagacaaag ccggattgcc cttatggccg ataatttcag
     2341 caagtcaaaa aaggattgga aagaacgatt tctccgactc agtaagaacg tgagtaagat
     2401 cccgcccttg agccatgata tgaaggtatg taaaaggcca aacgggaagt ttgtcctcca
     2461 gataccttgc gatccaattt acacccggca aatccaagtt cacacaagcg actctatttg
     2521 cagtatagat cctggcggca gaacattcgc cacttgttat gatccctcca atataaaagc
     2581 ctttcagatt ggtcctgaag cagataaaaa agaggtgatc cacaagtacc acgaaaaaat
     2641 tgactatgta caccggctcc tggcgtatgc gcagaagaaa aagcaaactc aggcggtaca
     2701 ggatcggatt gggcagctca aaaagctcca tcttaagctt aagacgtatg tcgacgatgt
     2761 gcatttgaaa ttgtgttcat accttgttaa aaactataaa ttggtagtgc tgggaaagat
     2821 ctcagtatct agtatcgtgc gcaaagaccg acccaatcac cttgctaaaa aagccaacag
     2881 ggaccttctt tgctggcagc actacaggtt tcggcagaga ttgctccaca gagtacgggg
     2941 aaccgactgc gaagcgattg cccaggacga aaggtacaca agtaaaacgt gtggcaactg
     3001 tggtgttaaa aataataagt tgggtggaaa ggaaacgttc atttgcgaat cttgcaatta
     3061 caagacgcat agggacgtga atggcgcaag gaacatcttg tgtaagtacc ttgggctgtt
     3121 tccgttcgca gccggaggtg ggggctctaa aaggccagct gctacgaaga aggccgggca
     3181 ggctaagaag aaaaaataac ctaggtcaga gctcgctgat cagcctcgac tgtgccttct
     3241 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3301 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3361 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3421 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     3481 ggctcgagat ccactagttc tagcctcgag gctagagcgg ccgccactgg ccgtcgtttt
     3541 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     3601 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     3661 gcgcagcctg aatggcgaat ggaaattgta agcgttaata ttttgttaaa attcgcgtta
     3721 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     3781 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     3841 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     3901 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     3961 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     4021 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     4081 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca
     4141 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     4201 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     4261 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     4321 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     4381 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     4441 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     4501 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     4561 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     4621 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     4681 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     4741 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     4801 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     4861 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     4921 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     4981 cgtggctctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     5041 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     5101 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     5161 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     5221 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     5281 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5341 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5401 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     5461 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5521 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5581 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5641 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     5701 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     5761 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     5821 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     5881 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     5941 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     6001 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     6061 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     6121 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     6181 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     6241 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgaggcg
     6301 cgccg
//
