LOCUS       sequence-388010-        6083 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector with Arabidopsis codon optimized Casphi-2 protein, 
            with SV40 NLS at the C-terminal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6083)
  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 6083)
  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_version2
FEATURES             Location/Qualifiers
     source          1..6083
                     /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)"
     intron          4202..4390
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     CDS             5709..5729
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5736..5756
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5775..5822
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     terminator      5831..6083
                     /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 tgccgaagcc cgccgtcgaa tcagagtttt ccaaagtcct caagaaacac tttcctgggg
     3301 agcgttttag gtctagctat atgaagaggg ggggtaaaat tctggcagca caaggcgagg
     3361 aagctgtagt ggcgtacttg cagggaaaga gtgaggagga accgccgaat tttcagccgc
     3421 cggcgaagtg ccacgtggtc accaaaagca gggatttcgc agaatggccc ataatgaaag
     3481 cctctgaagc catacagagg tacatctatg cgctcagcac tacagagcga gctgcctgca
     3541 aaccgggtaa gagctcagaa agtcacgcgg cctggttcgc ggctacaggg gtgagcaatc
     3601 acggctattc tcatgtacaa ggtcttaacc tgatctttga ccacacgcta ggacgatacg
     3661 atggcgtttt aaagaaagta cagcttcgaa atgagaaggc ccgagctaga ttggaaagca
     3721 taaacgcctc acgagctgat gaaggattgc cggaaatcaa ggccgaggag gaggaggtag
     3781 ctacaaatga aacaggtcat ctactacagc cgccaggcat aaacccatca ttctacgtct
     3841 accagaccat atctccgcag gcttaccgac caagggacga aatagtgtta ccacccgagt
     3901 acgccggtta cgtcagggat ccgaacgctc cgattccact gggcgtggtc aggaaccgtt
     3961 gtgacataca gaagggttgc ccgggatata tacccgagtg gcagagggaa gctggtacgg
     4021 caattagtcc caagacagga aaagcagtga cggttccagg acttagcccg aagaagaata
     4081 aacgtatgcg taggtactgg aggtcagaaa aggagaaggc tcaagatgca cttctcgtaa
     4141 ctgtaaggat aggtaccgat tgggtagtca tagacgtaag aggattacta agaaacgcca
     4201 ggtaagtttc tgcttctacc tttgatatat atataataat tatcattaat tagtagtaat
     4261 ataatatttc aaatattttt ttcaaaataa aagaatgtag tatatagcaa ttgcttttct
     4321 gtagtttata agtgtgtata ttttaattta taacttttct aatatatgac caaaatttgt
     4381 tgatgtgcag gtggaggact attgctccca aggacataag tttaaatgca cttctagatt
     4441 tatttaccgg tgatccagtc atcgatgtca gacgaaacat cgtgaccttc acctatacct
     4501 tggacgcttg cggaacttat gctagaaaat ggactctcaa gggaaaacag acaaaagcaa
     4561 ccttagataa actgacagcg acacaaactg tggccttagt tgctatagat ctgggacaaa
     4621 caaacccaat tagcgcgggt atcagtcgtg tcacacagga gaacggggcc ctccagtgcg
     4681 aaccgcttga tcgttttaca ttgcctgatg accttttgaa agatatttct gcgtaccgaa
     4741 ttgcatggga ccgtaacgag gaggaactca gggccagatc cgttgaggca ctcccagagg
     4801 cacaacaagc agaggtcaga gctctggacg gggtctccaa agagaccgcg cgtacacagt
     4861 tgtgcgcgga cttcggtctg gacccaaagc gactaccgtg ggataaaatg agtagcaata
     4921 ccacgttcat aagcgaggcg ctcctttcca acagcgtatc ccgtgaccaa gtattcttta
     4981 ccccggcccc aaagaaagga gccaagaaga aagcaccggt ggaagtgatg cgaaaagaca
     5041 ggacatgggc gcgagcgtac aaaccacgac tctcagtaga agcacaaaag ttgaaaaatg
     5101 aggctctttg ggctttgaag cgtacctctc cagaatatct aaagttgtca cgacgtaaag
     5161 aagaattgtg taggaggtcc attaattacg tgatagaaaa aactagaagg cgaacccaat
     5221 gccaaattgt gatccccgtt atagaagatc taaatgtccg atttttccac gggtctggca
     5281 aacgactccc gggctgggat aacttcttca cggcaaagaa ggaaaaccga tggtttatcc
     5341 aagggctaca taaggcgttt tctgacttaa ggacccaccg atccttctac gtgttcgagg
     5401 tgcgtccaga gagaacatca atcacctgtc cgaaatgcgg gcactgtgaa gtggggaaca
     5461 gggatggaga agcctttcag tgtctcagtt gcggtaagac atgcaacgca gatctggacg
     5521 tagcgacaca taacttgact caagtggcgc tcaccggcaa aacaatgccg aagagagagg
     5581 agcctagaga tgcacaaggg acagcccccg cgagaaagac gaagaaagct agcaagtcaa
     5641 aggcaccccc ggctgaacgt gaggatcaga ctcccgctca agagccgtcc caaacgtccg
     5701 gatccggacc gaagaaaaag cgaaaggtag aggatcctaa aaagaagcgt aaagtctcct
     5761 tgggttctgg ctccgactat aaggatgacg atgacaaaga ctataaggat gacgacgaca
     5821 agtgactgca gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc
     5881 cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg taataattaa
     5941 catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata
     6001 catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc
     6061 ggtgtcatct atgttactag atc
//
