LOCUS       sequence-449449-        7039 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid codes for the OgeuIscB nickase .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7039)
  AUTHORS   Mangin A, Dion V, Menzies G
  TITLE     Developing small Cas9 hybrids using molecular modeling
  JOURNAL   bioRxiv 2023.10.24.563270
REFERENCE   2  (bases 1 to 7039)
  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: "bioRxiv 
            2023.10.24.563270"
COMMENT     Sequence Label: pcDNA3.1-CMV-SV40NLS-OgeuIscBE193A-3XHA
FEATURES             Location/Qualifiers
     source          1..7039
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(9..800,801..869))
                     /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     632..651
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(870..974)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1049..1068)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1240..1619
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1620..1823
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1774..1794
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1868..1887
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1868..1886
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1922..1931
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1937..1957
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3455..3502
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3509..3589
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3635..3652)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3641..3865
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3911..4339
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3998..4017)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4208..4229
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4348..4368)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4353..4682
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4533..4668
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4595..4614
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4749..5543
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4803..4822)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5413..5432
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5717..5838
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5754..5773)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5808..5827
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5887..5903)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5887..5903)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5900..5922)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5911..5927
                     /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(5935..5941,5942..5959,5960..5965))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5980..6001
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6118..6135)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6289..6877)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6369..6388)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
       61 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
      121 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
      181 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
      241 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
      301 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
      361 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
      421 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
      481 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
      541 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
      601 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
      661 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
      721 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
      781 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
      841 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
      901 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
      961 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     1021 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     1081 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     1141 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
     1201 ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt
     1261 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1321 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1381 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1441 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     1501 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1561 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     1621 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     1681 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1741 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     1801 gggaggtcta tataagcaga gctctctggc taactagaga acccactgct tactggctta
     1861 tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct
     1921 tgccaccatg ggatctccaa aaaagaaacg gaaggtggga tctatggccg tggtctacgt
     1981 gatctctaag tccggcaaac cactgatgcc caccaccaga tgcggccacg tgcggatctt
     2041 gctcaaggaa ggcaaggcta gagtggtgga aagaaagccc ttcaccatcc agctgaccta
     2101 cgagtctgcc gaggaaaccc agcctctggt gctgggcatt gaccctggca gaaccaatat
     2161 cggtatgagc gtggtgaccg agagcggaga gagcgtgttc aacgcccaaa tcgagacaag
     2221 aaacaaggac gtgcctaagc tgatgaagga cagaaagcag tataggatgg cccacagaag
     2281 gctgaagcgg cggtgcaaaa gacggcggag agccaaggct gccggcaccg ccttcgagga
     2341 gggcgaaaag caaagactgc tgcctggctg cttcaagcct attacatgca agagcatccg
     2401 gaacaaggaa gccagattca acaaccgcaa gcggcctgtg ggatggctga cacctaccgc
     2461 caatcacctg ctggtcaccc acctgaacgt ggttaagaag gtgcagaaga tcctgcccgt
     2521 ggccaaggtg gtgctggccc tgaatcggtt cagctttatg gccatgaaca accccaaggt
     2581 tcagagatgg cagtaccagc ggggccctct gtacggcaaa ggcagcgtgg aagaggccgt
     2641 gtccatgcag caggatggac actgcctgtt ttgtaaacac ggcatcgacc actaccacca
     2701 cgtggtcccc agaagaaaga acggcagcga gacactggaa aacagagtgg gcctttgtga
     2761 agaacaccat agactggtgc acacagataa ggagtgggag gccaacctgg ccagcaagaa
     2821 gtctggaatg aacaagaaat accacgccct gtctgtgctg aaccagatca tcccctacct
     2881 ggctgatcag ctggccgata tgttccccgg aaatttctgc gtgacaagcg gccaagacac
     2941 ctatctgttt cgggaagagc atggcatccc taaggaccac tacctggacg cctactgcat
     3001 cgcctgcagc gcactgaccg acgccaagaa ggtgagcagc cctaagggca gaccatacat
     3061 ggtgcaccag ttcagaaggc atgatagaca ggcctgtcac aaggccaatc tgaaccggag
     3121 ctactacatg ggcggcaagc tcgttgccac caaccggcac aaagctatgg accagaaaac
     3181 tgacagcctg gaagagtaca gagccgctca cagcgccgct gacgtgtcta aactgaccgt
     3241 gaagcaccct tctgctcagt acaaggatat gagcagaatc atgcctggca gcatcctggt
     3301 gagcggcgag ggcaaactgt tcacactgag cagatctgag ggaagaaaca aaggccaggt
     3361 gaactacttc gtgtccaccg agggcatcaa gtactgggcc agaaagtgcc agtatctgcg
     3421 gaacaacggc ggactgcaga tctacgtggg atccaaaagg ccggcggcca cgaaaaaggc
     3481 cggccaggca aaaaagaaaa agggatctta cccatacgat gttccagatt acgcttaccc
     3541 atacgatgtt ccagattacg cttacccata cgatgttcca gattacgctt gaggccgctc
     3601 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     3661 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3721 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3781 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     3841 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     3901 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3961 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4021 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4081 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     4141 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4201 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     4261 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     4321 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     4381 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     4441 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4501 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4561 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4621 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     4681 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     4741 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4801 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4861 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4921 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4981 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     5041 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     5101 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     5161 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     5221 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     5281 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     5341 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     5401 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     5461 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     5521 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     5581 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     5641 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     5701 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     5761 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     5821 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     5881 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5941 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     6001 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     6061 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     6121 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     6181 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     6241 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6301 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6361 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     6421 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     6481 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     6541 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6601 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6661 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6721 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6781 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6841 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6901 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6961 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     7021 tatatatgag taaacttgg
//
