LOCUS       sequence-454042-        5901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of EVOLVEpro PsaCas12f (epPsaCas12f).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5901)
  AUTHORS   Jiang K, Yan Z, Di Bernardo M, Sgrizzi SR, Villiger L, Kayabolen A, 
            Kim B, Carscadden JK, Hiraizumi M, Nishimasu H, Gootenberg JS, 
            Abudayyeh OO
  TITLE     Rapid in silico directed evolution by a protein language model with 
            EVOLVEpro
  JOURNAL   Science 0, eadr6006
  PUBMED    39071429
REFERENCE   2  (bases 1 to 5901)
  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: "Science 0, 
            eadr6006"
COMMENT     Sequence Label: pDF850
FEATURES             Location/Qualifiers
     source          1..5901
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(70..89)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        261..640
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        641..844
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     795..815
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     886..905
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        886..904
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             954..974
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2775..2795
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2805..2861
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2862..3419
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGGD"
     CDS             3425..3442
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3465..3482)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3471..3695
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3766..3782)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3766..3782)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3779..3801)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3790..3806
                     /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(3814..3820,3821..3838,3839..3844))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3859..3880
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3997..4014)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4168..4756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4248..4267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4927..5718,5719..5787))
                     /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     5550..5569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5788..5892)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccgaaaagtg ccacctgacg tcgacggatc gggagatcga tctcccgatc ccctagggtc
       61 tactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg
      121 tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt
      181 gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt
      241 acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac
      301 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaattg
      361 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      421 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      481 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      541 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      601 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      661 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
      721 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
      781 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
      841 agctggttta gtgaaccgtc agatccgcta gagatccgcg gccgctaata cgactcacta
      901 tagggagagc cgccaccatg aaacggacag ccgacggaag cgagttcgag tcaccaaaga
      961 agaagcggaa agtcatgccc agtgagacat acatcaccaa gactctcagt ctgaagctca
     1021 tcccttctga cgaggagaag caggctttgg aaaactactt tattaccttc caacgggccg
     1081 taaatttcgc cattgatcga atagtggata tacgttcctc tttcagatat ctgaataaga
     1141 atgagcagtt cccagcggtc tgtgattgtt gtggcaagaa ggagaaaatt atgtacgtga
     1201 acatatctaa caaaaccttt aaattcaagc caagccgaaa ccagaaggat cgatacacta
     1261 aagatattta caccatcaag ccaaacgctc acatttgtaa aacttgttac agcggagtcg
     1321 ccgggaacat gtttatccgc aaacagatgt acccaaacga taaagaagga tggaaagttt
     1381 ctcgctccta taacataaaa gtcaatgctc cagggttgac tggcaccgaa tatgcgatgg
     1441 caattcggaa ggcaatcagt atactgaggt cttttgaaaa gcgccgcagg aatgcagaac
     1501 gcagagcaat tgaatacgag aaatcaaaga aagagtactt ggagctcatt gacgatgtgg
     1561 agaagggaaa gactaacaaa atcgttgtcc ttgaaaagga aggccaccaa cgggttaaac
     1621 gttataaaca taagaactgg cccgaaaagt ggcaaggaat ttcactgaac aaagccaaaa
     1681 gcaaagtaaa ggatatcgag aaaaggatta agaagctgaa agaatggaaa cacccaacac
     1741 tgaatcgtcc atatgttgaa ctccataaga ataacgtgcg aatcgtaggc tatgagactg
     1801 ttgagctcaa actgggaaat aagatgtaca caatccattt cgcatcaata agtaacttga
     1861 gaaagccatt tcggaagcaa aagaagaagt ccattgaata tctcaagcat ttgctcacac
     1921 tcgccctcaa gagaaatctg gaaacgtatc cctcaattat caagagaggc gttaacttct
     1981 tcttgcaata ccctgttcgt gtaactgtaa aggtccctaa actgacaaag aactttaagg
     2041 ccttcggaat cgatagaggc gttaaccgcc tcgctgtagg atgtatcata tccaaggacg
     2101 gaaaactgac caacaagaat atcttcttct ttcacggtaa agaggcgtgg gccaaagaaa
     2161 accgatacaa gaaaatccgc gatcgcctct acgctatggc gaagaaactc cggggagata
     2221 aaacgaagaa aattcgtctg tatcacgaaa ttcgcaagaa gttccgacac aaagtcaaat
     2281 atttcaggcg gaattacctt cacaatataa gcaaacaaat agttgagatc gctcccgaga
     2341 acactcctac tgtgattgtg cttgaggatc tgcggtacct gagggaacgg acctataggg
     2401 gaaaaggccg cagcaagaag gccaagaaaa caaattacaa actcaatact ttcacctacc
     2461 gaatgctgat tgatatgatt aaatacaagg cagaagaggc gggagtacct gttatgatca
     2521 ttgatccacg gaatacatcc agaaaatgca gtaaatgcgg atatgtcgac gagaacaacc
     2581 gcaaacaggc atcttttaag tgccttaagt gtggatattc tttgaacgca gacttgaatg
     2641 ccgcagtgaa cattgcaaaa gccttttatg aatgccctac ttttcgatgg gaagaaaagc
     2701 tgcacgcgta tgtctgcagc gaacccgaca aatctggcgg ctcaaaaaga accgccgacg
     2761 gcagcgaatt cgagcccaag aagaagagga aagtcggaag cggagctact aacttcagcc
     2821 tgctgaagca ggctggcgac gtggaggaga accctggacc tatgggagtc aaagttctgt
     2881 ttgccctgat ctgcatcgct gtggccgagg ccaagcccac cgagaacaac gaagacttca
     2941 acatcgtggc cgtggccagc aacttcgcga ccacggatct cgatgctgac cgcgggaagt
     3001 tgcccggcaa gaagctgccg ctggaggtgc tcaaagagat ggaagccaat gcccggaaag
     3061 ctggctgcac caggggctgt ctgatctgcc tgtcccacat caagtgcacg cccaagatga
     3121 agaagttcat cccaggacgc tgccacacct acgaaggcga caaagagtcc gcacagggcg
     3181 gcataggcga ggcgatcgtc gacattcctg agattcctgg gttcaaggac ttggagccca
     3241 tggagcagtt catcgcacag gtcgatctgt gtgtggactg cacaactggc tgcctcaaag
     3301 ggcttgccaa cgtgcagtgt tctgacctgc tcaagaagtg gctgccgcaa cgctgtgcga
     3361 cctttgccag caagatccag ggccaggtgg acaagatcaa gggggccggt ggtgactaac
     3421 cggtcatcat caccatcacc attgagttta aacccgctga tcagcctcga ctgtgccttc
     3481 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3541 cactcccact gtcctttcct aataaaatga gaaaattgca tcgcattgtc tgagtaggtg
     3601 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3661 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3721 gggctcgata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     3781 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3841 taaagcctag ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3901 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3961 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4021 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4081 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4141 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4201 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4261 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4321 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4381 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4441 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4501 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4561 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4621 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4681 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4741 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgaca ctcagtggaa
     4801 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4861 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4921 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4981 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5041 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5101 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5161 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5221 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5281 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5341 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5401 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5461 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5521 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5581 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5641 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5701 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5761 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5821 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5881 aggggttccg cgcacatttc c
//
