LOCUS       sequence-479781-        6937 bp    DNA     circular SYN 24-JUL-2025
DEFINITION  Expresses the Wild Type Francisella novicida cas12a gene and a NT 
            crRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6937)
  AUTHORS   Della Valle S, Orsi E, Creutzburg SCA, Jansen LFM, Pentari E, Beisel
            CL, Steel H, Nikel PI, Staals RHJ, Claassens NJ, van der Oost J, 
            Huang WE, Patinios C
  TITLE     Streamlined and efficient genome editing in Cupriavidus necator H16 
            using an optimised SIBR-Cas system
  JOURNAL   bioRxiv 2024.11.24.625072; doi: 10.1101/2024.11.24.625072
REFERENCE   2  (bases 1 to 6937)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 24, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.11.24.625072; doi: 10.1101/2024.11.24.625072"
COMMENT     Sequence Label: pCas12a-NT
FEATURES             Location/Qualifiers
     source          1..6937
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(3808..3813,3814..3831,3832..3838)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     primer_bind     complement(4092..4111)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4103..4918
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4989..5651)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      5652..6423
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     terminator      complement(6538..6624)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     6639..6661
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     promoter        join(6668..6673,6674..6691,6692..6698)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
ORIGIN
        1 atcagttttt tatagaggag atattaagtt cggtttgtat tagcgaagat ttattacaaa
       61 actattctga tgtttatttt aaacttaaaa agagtgatga tgataatcta caaaaagatt
      121 ttaaaagtgc aaaagatacg ataaagaaac aaatatctga atatataaag gactcagaga
      181 aatttaagaa tttgtttaat caaaacctta tcgatgctaa aaaagggcaa gagtcagatt
      241 taattctatg gctaaagcaa tctaaggata atggtataga actatttaaa gccaatagtg
      301 atatcacaga tatagatgag gcgttagaaa taatcaaatc ttttaaaggt tggacaactt
      361 attttaaggg ttttcatgaa aatagaaaaa atgtttatag tagcaatgat attcctacat
      421 ctattattta taggatagta gatgataatt tgcctaaatt tctagaaaat aaagctaagt
      481 atgagagttt aaaagacaaa gctccagaag ctataaacta tgaacaaatt aaaaaagatt
      541 tggcagaaga gctaaccttt gatattgact acaaaacatc tgaagttaat caaagagttt
      601 tttcacttga tgaagttttt gagatagcaa actttaataa ttatctaaat caaagtggta
      661 ttactaaatt taatactatt attggtggta aatttgtaaa tggtgaaaat acaaagagaa
      721 aaggtataaa tgaatatata aatctatact cacagcaaat aaatgataaa acactcaaaa
      781 aatataaaat gagtgtttta tttaagcaaa ttttaagtga tacagaatct aaatcttttg
      841 taattgataa gttagaagat gatagtgatg tagttacaac gatgcaaagt ttttatgagc
      901 aaatagcagc ttttaaaaca gtagaagaaa aatctattaa agaaacacta tctttattat
      961 ttgatgattt aaaagctcaa aaacttgatt tgagtaaaat ttattttaaa aatgataaat
     1021 ctcttactga tctatcacaa caagtttttg atgattatag tgttattggt acagcggtac
     1081 tagaatatat aactcaacaa atagcaccta aaaatcttga taaccctagt aagaaagagc
     1141 aagaattaat agccaaaaaa actgaaaaag caaaatactt atctctagaa actataaagc
     1201 ttgccttaga agaatttaat aagcatagag atatagataa acagtgtagg tttgaagaaa
     1261 tacttgcaaa ctttgcggct attccgatga tatttgatga aatagctcaa aacaaagaca
     1321 atttggcaca gatatctatc aaatatcaaa atcaaggtaa aaaagaccta cttcaagcta
     1381 gtgcggaaga tgatgttaaa gctatcaagg atcttttaga tcaaactaat aatctcttac
     1441 ataaactaaa aatatttcat attagtcagt cagaagataa ggcaaatatt ttagacaagg
     1501 atgagcattt ttatctagta tttgaggagt gctactttga gctagcgaat atagtgcctc
     1561 tttataacaa aattagaaac tatataactc aaaagccata tagtgatgag aaatttaagc
     1621 tcaattttga gaactcgact ttggctaatg gttgggataa aaataaagag cctgacaata
     1681 cggcaatttt atttatcaaa gatgataaat attatctggg tgtgatgaat aagaaaaata
     1741 acaaaatatt tgatgataaa gctatcaaag aaaataaagg cgagggttat aaaaaaattg
     1801 tttataaact tttacctggc gcaaataaaa tgttacctaa ggttttcttt tctgctaaat
     1861 ctataaaatt ttataatcct agtgaagata tacttagaat aagaaatcat tccacacata
     1921 caaaaaatgg tagtcctcaa aaaggatatg aaaaatttga gtttaatatt gaagattgcc
     1981 gaaaatttat agatttttat aaacagtcta taagtaagca tccggagtgg aaagattttg
     2041 gatttagatt ttctgatact caaagatata attctataga tgaattttat agagaagttg
     2101 aaaatcaagg ctacaaacta acttttgaaa atatatcaga gagctatatt gatagcgtag
     2161 ttaatcaggg taaattgtac ctattccaaa tctataataa agatttttca gcttatagca
     2221 aagggcgacc aaatctacat actttatatt ggaaagcgct gtttgatgag agaaatcttc
     2281 aagatgtggt ttataagcta aatggtgagg cagagctttt ttatcgtaaa caatcaatac
     2341 ctaaaaaaat cactcaccca gctaaagagg caatagctaa taaaaacaaa gataatccta
     2401 aaaaagagag tgtttttgaa tatgatttaa tcaaagataa acgctttact gaagataagt
     2461 ttttctttca ctgtcctatt acaatcaatt ttaaatctag tggagctaat aagtttaatg
     2521 atgaaatcaa tttattgcta aaagaaaaag caaatgatgt tcatatatta agtatagata
     2581 gaggtgaaag acatttagct tactatactt tggtagatgg taaaggcaat atcatcaaac
     2641 aagatacttt caacatcatt ggtaatgata gaatgaaaac aaactaccat gataagcttg
     2701 ctgcaataga gaaagatagg gattcagcta ggaaagactg gaaaaagata aataacatca
     2761 aagagatgaa agagggctat ctatctcagg tagttcatga aatagctaag ctagttatag
     2821 agtataatgc tattgtggtt tttgaggatt taaattttgg atttaaaaga gggcgtttca
     2881 aggtagagaa gcaggtctat caaaagttag aaaaaatgct aattgagaaa ctaaactatc
     2941 tagttttcaa agataatgag tttgataaaa ctgggggagt gcttagagct tatcagctaa
     3001 cagcaccttt tgagactttt aaaaagatgg gtaaacaaac aggtattatc tactatgtac
     3061 cagctggttt tacttcaaaa atttgtcctg taactggttt tgtaaatcag ttatatccta
     3121 agtatgaaag tgtcagcaaa tctcaagagt tctttagtaa gtttgacaag atttgttata
     3181 accttgataa gggctatttt gagtttagtt ttgattataa aaactttggt gacaaggctg
     3241 ccaaaggcaa gtggactata gctagctttg ggagtagatt gattaacttt agaaattcag
     3301 ataaaaatca taattgggat actcgagaag tttatccaac taaagagttg gagaaattgc
     3361 taaaagatta ttctatcgaa tatgggcatg gcgaatgtat caaagcagct atttgcggtg
     3421 agagcgacaa aaagtttttt gctaagctaa ctagtgtcct aaatactatc ttacaaatgc
     3481 gtaactcaaa aacaggtact gagttagatt atctaatttc accagtagca gatgtaaatg
     3541 gcaatttctt tgattcgcga caggcgccaa aaaatatgcc tcaagatgct gatgccaatg
     3601 gtgcttatca tattgggcta aaaggtctga tgctactagg taggatcaaa aataatcaag
     3661 agggcaaaaa actcaatttg gttatcaaaa atgaagagta ttttgagttc gtgcagaata
     3721 ggaataacta agcggccgca taatgatgtg ttatcattga tgcgaggtcg cctatacctc
     3781 cccgcttcgg cggggttttt tcccgggttt acactttatg cttccggctc gtataatgtg
     3841 tggtctaaga actttaaata atttctactg ttgtagatga gtcttcctga gtgacgagca
     3901 acgagactgg aagacatgtc taagaacttt aaataatttc tactgttgta gatagatacc
     3961 ggacaacgtg tctccccgct tcggcggggt tttttcctag gttgtgtctc aaaatctctg
     4021 atgttacatt gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat
     4081 aaacagtaat acaaggggtg ttatgagcca tattcagcgt gaaacgagct gtagccgtcc
     4141 gcgtctgaac agcaacatgg atgcggatct gtatggctat aaatgggcgc gtgataacgt
     4201 gggtcagagc ggcgcgacca tttatcgtct gtatggcaaa ccggatgcgc cggaactgtt
     4261 tctgaaacat ggcaaaggca gcgtggcgaa cgatgtgacc gatgaaatgg tgcgtctgaa
     4321 ctggctgacc gaatttatgc cgctgccgac cattaaacat tttattcgca ccccggatga
     4381 tgcgtggctg ctgaccaccg cgattccggg caaaaccgcg tttcaggtgc tggaagaata
     4441 tccggatagc ggcgaaaaca ttgtggatgc gctggccgtg tttctgcgtc gtctgcatag
     4501 cattccggtg tgcaactgcc cgtttaacag cgatcgtgtg tttcgtctgg cccaggcgca
     4561 gagccgtatg aacaacggcc tggtggatgc gagcgatttt gatgatgaac gtaacggctg
     4621 gccggtggaa caggtgtgga aagaaatgca taaactgctg ccgtttagcc cggatagcgt
     4681 ggtgacccac ggcgatttta gcctggataa cctgattttc gatgaaggca aactgattgg
     4741 ctgcattgat gtgggccgtg tgggcattgc ggatcgttat caggatctgg ccattctgtg
     4801 gaactgcctg ggcgaattta gcccgagcct gcaaaaacgt ctgtttcaga aatatggcat
     4861 tgataatccg gatatgaaca aactgcaatt tcatctgatg ctggatgaat ttttctaata
     4921 attaattgga ccgcggtccg agagacgcat ggcagtatct cagactgtgt atcggcacgt
     4981 ctcactactt accggcgcgg cagcgttacc cgtgtcggcg gctccaacgg ctcgccatcg
     5041 tccagaaaac acggctcatc gggcatcggc aggcgctgct gcccgcgccg ttcccattcc
     5101 tccgtttcgg tcaaggctgg caggtctggt tccatgcccg gaatgccggg ctggctgggc
     5161 ggctcctcgc cggggccggt cggtagttgc tgctcgcccg gatacagggt cgggatgcgg
     5221 cgcaggtcgc catgccccaa cagcgattcg tcctggtcgt cgtgatcaac caccacggcg
     5281 gcactgaaca ccgacaggcg caactggtcg cggggctggc cccacgccac gcggtcattg
     5341 accacgtagg ccgacacggt gccggggccg ttgagcttca cgacggagat ccagcgctcg
     5401 gccaccaagt ccttgactgc gtattggacc gtccgcaaag aacgtccgat gagcttggaa
     5461 agtgttttct ggctgaccac cacggcgttc tggtggccca tctgcgccac gaggtgatgc
     5521 agcagcattg ccgccgtggg tttcctcgca ataagcccgg cccacgcctc atgcgctttg
     5581 cgttccgttt gcacccagtg accgggcttg ttcttggctt gaatgccgat ttctctggac
     5641 tgcgtggcca tgcttatctc catgcggtag gggtgccgca cggttgcggc accatgcgca
     5701 atcagctgca acttttcggc agcgcgacaa caattatgcg ttgcgtaaaa gtggcagtca
     5761 attacagatt ttctttaacc tacgcaatga gctattgcgg ggggtgccgc aatgagctgt
     5821 tgcgtacccc ccttttttaa gttgttgatt tttaagtctt tcgcatttcg ccctatatct
     5881 agttctttgg tgcccaaaga agggcacccc tgcggggttc ccccacgcct tcggcgcggc
     5941 tccccctccg gcaaaaagtg gcccctccgg ggcttgttga tcgactgcgc ggccttcggc
     6001 cttgcccaag gtggcgctgc ccccttggaa cccccgcact cgccgccgtg aggctcgggg
     6061 ggcaggcggg cgggcttcgc ccttcgactg cccccactcg cataggcttg ggtcgttcca
     6121 ggcgcgtcaa ggccaagccg ctgcgcggtc gctgcgcgag ccttgacccg ccttccactt
     6181 ggtgtccaac cggcaagcga agcgcgcagg ccgcaggccg gaggcttttc cccagagaaa
     6241 attaaaaaaa ttgatggggc aaggccgcag gccgcgcagt tggagccggt gggtatgtgg
     6301 tcgaaggctg ggtagccggt gggcaatccc tgtggtcaag ctcgtgggca ggcgcagcct
     6361 gtccatcagc ttgtccagca gggttgtcca cgggccgagc gaagcgagcc agccggtggc
     6421 cgctcgcggc catcgtccac atatccacgg gctggcaagg gagcgcagcg accgcgcagg
     6481 gcgaagcccg gagagcaagc ccgtaggggg ggcgcgccca gctgtctagg gcggcggatt
     6541 tgtcctactc aggagagcgt tcaccgacaa acaacagata aaacgaaagg cccagtcttt
     6601 cgactgagcc tttcgtttta tttgatgcct ttaattaaag cggataacaa tttcacacag
     6661 gaggccgttt acactttatg cttccggctc gtataatgtg tggggaaacc acaacggttt
     6721 ccctctagaa ataattttgt ttaactataa gaaggagata tacatatgtc aatttatcaa
     6781 gaatttgtta ataaatatag tttaagtaaa actctaagat ttgagttaat cccacagggt
     6841 aaaacacttg aaaacataaa agcaagaggt ttgattttag atgatgagaa aagagctaaa
     6901 gactacaaaa aggctaaaca aataattgat aaatatc
//
