LOCUS       sequence-388007-        6110 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector with Arabidopsis codon optimized Casphi-2 protein, 
            with SV40 NLS at the N-terminal and nucleoplasmin NLS at the 
            C-terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6110)
  AUTHORS   Li Z, Zhong Z, Wu Z, Pausch P, Al-Shayeb B, Amerasekera J, Doudna 
            JA, Jacobsen SE
  TITLE     Genome editing in plants using the compact editor CasPhi.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Jan 24;120(4):e2216822120. doi: 
            10.1073/pnas.2216822120. Epub 2023 Jan 18.
  PUBMED    36652483
REFERENCE   2  (bases 1 to 6110)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-01-24"; volume: "120(4):e2216822120. 
            doi"; pages: " 10.1073/pnas.2216822120. Epub 2023 Jan 18"
COMMENT     Sequence Label: HBT_pcoCASphi_version1
FEATURES             Location/Qualifiers
     source          1..6110
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..55)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(39..55)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(52..74)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    63..79
                     /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(87..93,94..111,112..117))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    132..153
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(270..287)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(441..1029)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(521..540)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1200..1991,1992..2060))
                     /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     1823..1842
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2061..2165)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2233..2251
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2289..2311)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2411..2430
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2624..2646
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2638..2655
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2639..2655
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        join(2722..3020,3021..3043,3044..3231)
                     /label=35SPPDK promoter
                     /note="hybrid promoter consisting of the cauliflower mosaic
                     virus 35S enhancer fused to the maize C4PPDK basal promoter
                     (Yoo et al., 2007)"
     CDS             3243..3290
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             3297..3317
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          4304..4492
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     CDS             5802..5849
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     terminator      5858..6110
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
ORIGIN
        1 cgatgataag ctgtcaaaca tgagaattcg taatcatggt catagctgtt tcctgtgtga
       61 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
      121 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
      181 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
      241 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
      301 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
      361 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
      421 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
      481 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
      541 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
      601 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
      661 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
      721 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
      781 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
      841 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
      901 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
      961 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1021 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     1081 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     1141 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     1201 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     1261 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     1321 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     1381 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     1441 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     1501 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     1561 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     1621 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     1681 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     1741 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     1801 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     1861 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     1921 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     1981 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     2041 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     2101 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     2161 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     2221 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     2281 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     2341 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     2401 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
     2461 ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc
     2521 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     2581 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
     2641 aaaacgacgg ccagtgccaa gctctcgaga agcttactcc aagaatatca aagatacagt
     2701 ctcagaagac caaagggcta ttgagacttt tcaacaaagg gtaatatcgg gaaacctcct
     2761 cggattccat tgcccagcta tctgtcactt catcaaaagg acagtagaaa aggaaggtgg
     2821 cacctacaaa tgccatcatt gcgataaagg aaaggctatc gttcaagatg cctctgccga
     2881 cagtggtccc aaagatggac ccccacccac aaggagcatc gtggaaaaag aagacgttcc
     2941 aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa gggatgacgc
     3001 acaatcccac tatccttcgc cccaagcttg ggcccaagct tgggtcgcgc cccacggatg
     3061 gtataagaat aaaggcattc cgcgtgcagg attcacccgt tcgcctctca ccttttcgct
     3121 gtactctctc gccacacaca ccccctctcc agctccgttg gagctccgga cagcagcagg
     3181 cgcggggcgg tcacgtagta agcagctctc ggctccctct ccccttgctc cgtggatcca
     3241 tggattacaa ggatgatgat gataaggatt acaaggatga tgatgataag atggctccaa
     3301 agaagaagag aaaggttgga atccacggag ttccagctgc tatgccgaag cccgccgtcg
     3361 aatcagagtt ttccaaagtc ctcaagaaac actttcctgg ggagcgtttt aggtctagct
     3421 atatgaagag ggggggtaaa attctggcag cacaaggcga ggaagctgta gtggcgtact
     3481 tgcagggaaa gagtgaggag gaaccgccga attttcagcc gccggcgaag tgccacgtgg
     3541 tcaccaaaag cagggatttc gcagaatggc ccataatgaa agcctctgaa gccatacaga
     3601 ggtacatcta tgcgctcagc actacagagc gagctgcctg caaaccgggt aagagctcag
     3661 aaagtcacgc ggcctggttc gcggctacag gggtgagcaa tcacggctat tctcatgtac
     3721 aaggtcttaa cctgatcttt gaccacacgc taggacgata cgatggcgtt ttaaagaaag
     3781 tacagcttcg aaatgagaag gcccgagcta gattggaaag cataaacgcc tcacgagctg
     3841 atgaaggatt gccggaaatc aaggccgagg aggaggaggt agctacaaat gaaacaggtc
     3901 atctactaca gccgccaggc ataaacccat cattctacgt ctaccagacc atatctccgc
     3961 aggcttaccg accaagggac gaaatagtgt taccacccga gtacgccggt tacgtcaggg
     4021 atccgaacgc tccgattcca ctgggcgtgg tcaggaaccg ttgtgacata cagaagggtt
     4081 gcccgggata tatacccgag tggcagaggg aagctggtac ggcaattagt cccaagacag
     4141 gaaaagcagt gacggttcca ggacttagcc cgaagaagaa taaacgtatg cgtaggtact
     4201 ggaggtcaga aaaggagaag gctcaagatg cacttctcgt aactgtaagg ataggtaccg
     4261 attgggtagt catagacgta agaggattac taagaaacgc caggtaagtt tctgcttcta
     4321 cctttgatat atatataata attatcatta attagtagta atataatatt tcaaatattt
     4381 ttttcaaaat aaaagaatgt agtatatagc aattgctttt ctgtagttta taagtgtgta
     4441 tattttaatt tataactttt ctaatatatg accaaaattt gttgatgtgc aggtggagga
     4501 ctattgctcc caaggacata agtttaaatg cacttctaga tttatttacc ggtgatccag
     4561 tcatcgatgt cagacgaaac atcgtgacct tcacctatac cttggacgct tgcggaactt
     4621 atgctagaaa atggactctc aagggaaaac agacaaaagc aaccttagat aaactgacag
     4681 cgacacaaac tgtggcctta gttgctatag atctgggaca aacaaaccca attagcgcgg
     4741 gtatcagtcg tgtcacacag gagaacgggg ccctccagtg cgaaccgctt gatcgtttta
     4801 cattgcctga tgaccttttg aaagatattt ctgcgtaccg aattgcatgg gaccgtaacg
     4861 aggaggaact cagggccaga tccgttgagg cactcccaga ggcacaacaa gcagaggtca
     4921 gagctctgga cggggtctcc aaagagaccg cgcgtacaca gttgtgcgcg gacttcggtc
     4981 tggacccaaa gcgactaccg tgggataaaa tgagtagcaa taccacgttc ataagcgagg
     5041 cgctcctttc caacagcgta tcccgtgacc aagtattctt taccccggcc ccaaagaaag
     5101 gagccaagaa gaaagcaccg gtggaagtga tgcgaaaaga caggacatgg gcgcgagcgt
     5161 acaaaccacg actctcagta gaagcacaaa agttgaaaaa tgaggctctt tgggctttga
     5221 agcgtacctc tccagaatat ctaaagttgt cacgacgtaa agaagaattg tgtaggaggt
     5281 ccattaatta cgtgatagaa aaaactagaa ggcgaaccca atgccaaatt gtgatccccg
     5341 ttatagaaga tctaaatgtc cgatttttcc acgggtctgg caaacgactc ccgggctggg
     5401 ataacttctt cacggcaaag aaggaaaacc gatggtttat ccaagggcta cataaggcgt
     5461 tttctgactt aaggacccac cgatccttct acgtgttcga ggtgcgtcca gagagaacat
     5521 caatcacctg tccgaaatgc gggcactgtg aagtggggaa cagggatgga gaagcctttc
     5581 agtgtctcag ttgcggtaag acatgcaacg cagatctgga cgtagcgaca cataacttga
     5641 ctcaagtggc gctcaccggc aaaacaatgc cgaagagaga ggagcctaga gatgcacaag
     5701 ggacagcccc cgcgagaaag acgaagaaag ctagcaagtc aaaggcaccc ccggctgaac
     5761 gtgaggatca gactcccgct caagagccgt cccaaacgtc caagagacca gctgctacca
     5821 agaaggctgg acaggctaag aagaagaagt gactgcagat cgttcaaaca tttggcaata
     5881 aagtttctta agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt
     5941 gaattacgtt aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt
     6001 ttttatgatt agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg
     6061 cgcaaactag gataaattat cgcgcgcggt gtcatctatg ttactagatc
//
