LOCUS       sequence-429629-        4944 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of BaFnuc for IVTT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4944)
  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 4944)
  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: pKJ224
FEATURES             Location/Qualifiers
     source          1..4944
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     80..99
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        80..98
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             130..152
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             171..188
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1707..1725)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1721..1768
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2134..2139,2140..2156,2157..2162))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2183..2199)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2183..2199)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2196..2218)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2207..2223
                     /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(2231..2237,2238..2255,2256..2261))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2276..2297
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2414..2431)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2585..3173)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2665..2684)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3344..4135,4136..4204))
                     /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     3967..3986
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4205..4309)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4377..4395
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4433..4455)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4555..4574
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4770..4792
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4784..4801
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4785..4801
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4921..4940
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
       61 gatctcgatc ccgcgaaatt aatacgactc actataggga gaccacaacg gtttccctct
      121 agaaataatt ttgtttaact ttaagaagga gatataccat ggggtcatcc caccatcatc
      181 atcatcactc tagtggcatg aagcgcacat acagcgctac gaaaagttcg ttaacacttt
      241 ggactgctgc ctccgtgaaa accacatcgg cgccaaaggt cgttaccact ttcagtggtt
      301 ggatgaaaaa aatcctgcca acacgggcag aaacttcact tacactgatt aaccctgcag
      361 atatcgcaga tccctcgccg ccgaagaaaa aagccaagaa aacgacgcca gcgactccaa
      421 agccaacttt acggatatat aaaattggtt tgagaccttc accggcccag cggaaaacgt
      481 tgaatgcgtg tatagtagct gcaaactttg cgtacaacca atgtgtgcac cttgtacaac
      541 acaaggtatg caaaccgcat ttatatgatc ttcaaaagat agtcgccaag atgaaaactc
      601 cggaagatat aaaccataga tacgcaccag accgggacgg ctggttttgg aagtcttcga
      661 cgatcgtacg cttgcttgca accaaagact tctgcgcagc ctacaaggcc attgtgtcca
      721 ataagaaaaa ggacgtagca gttataaaat acaaaacgta cgacgacccg gaagctataa
      781 atcccttatc aggtctgttc ggctgccaga agcagtacgc cacggtgaca caggcggggc
      841 ttagactgtt gccccgcctt tttggaaaag accctattcc tttagttaaa aagaagctta
      901 aagtcgcgac tatcgatcac gatttcaaaa ttgagaaaac ctctaagggg aagttcgtct
      961 tatgtcttac cgtagaatgt agcctgcttc gtcgggtcaa gccacccgca ccactgtttg
     1021 aggatgggta tattcacgct tgcggtatag atccaggtgt acgcagcttt gtgactgttt
     1081 atgatcctac gcgccaggat tgttaccagt ttggtacatc tgcgcaaaaa gccgaacgcc
     1141 ttgatcccat tacaaacgcg atcgacaatt ggaactcgtt tgtggaccaa caccgtgaca
     1201 aagcccctcc aactgcaatt gagtcttggt cccggaaaac aaagaagtta tggtacaagt
     1261 tgaagaacca ggtccggtct ttacacgatc aggtcatcgc acatctgttg ggcgcgtata
     1321 atttcatcag ccttggtaaa cttgatgtct cttgctttcg cagaggtaca acagcaaaga
     1381 gcacgaatcg ttggttacgt atttatcggc actttgagtt tcgcaccaaa ttgttagccc
     1441 gcgtcgaggg taccgataat tgtcgtgtag agatcaccga cgaacggtgg acatccaaaa
     1501 cctgtggcat gtgccgttcg atacacagag agcttggggc gaaagaattg ttcgagtgcc
     1561 cgaattgcca ttacacctgc catcgtgacg tacatggtgc acgcaatatt ctgttaagaa
     1621 gttttggtca gtttcccgta taactcgagc cttaggagat ccggctgcta acaaagcccg
     1681 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     1741 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatatccaca
     1801 ggacgggtgt ggtcgccatg atcgcgtagt cgatagtggc tccaagtagc gaagcgagca
     1861 ggactgggcg gcggccaaag cggtcggaca gtgctccgag aacgggtgcg catagaaatt
     1921 gcatcaacgc atatagcgct agcagcacgc catagtgact ggcgatgctg tcggaatgga
     1981 cgatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca
     2041 gggtgacggt gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac
     2101 tgcgttagca atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc
     2161 aaacatgaga attcgtaatc atgtcatagc tgtttcctgt gtgaaattgt tatccgctca
     2221 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     2281 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     2341 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     2401 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2461 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     2521 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2581 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     2641 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     2701 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2761 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2821 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2881 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2941 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3001 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     3061 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3121 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3181 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3241 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3301 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3361 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3421 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3481 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3541 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     3601 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     3661 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3721 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3781 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3841 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3901 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3961 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4021 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4081 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4141 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4201 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4261 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4321 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     4381 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
     4441 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     4501 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     4561 cagagcagat tgtactgaga gtgcaccata tatgcggtgt gaaataccgc acagatgcgt
     4621 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg
     4681 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     4741 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     4801 tgccaagctt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     4861 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     4921 ggtgatgtcg gcgatatagg cgcc
//
