LOCUS       sequence-467882-        5640 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Engineered human-codon optimized PseCas8 in mammalian cells (for 
            evoCAST).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5640)
  AUTHORS   Witte IP, Lampe GD, Eitzinger S, Miller SM, Berrios KN, McElroy AN, 
            King RT, Stringham OG, Gelsinger DR, Vo PLH, Chen AT, Tolar J, 
            Osborn MJ, Sternberg SH, Liu DR
  TITLE     Programmable gene insertion in human cells with a laboratory-evolved
            CRISPR-associated transposase.
  JOURNAL   Science. 2025 May 15;388(6748):eadt5199. doi: 
            10.1126/science.adt5199. Epub 2025 May 15.
  PUBMED    40373119
REFERENCE   2  (bases 1 to 5640)
  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."; 
            date: "2025-05-15"; volume: "388(6748):eadt5199. doi"; pages: " 
            10.1126/science.adt5199. Epub 2025 May 15"
COMMENT     Sequence Label: pCMV-evoCAST-eCas8
FEATURES             Location/Qualifiers
     source          1..5640
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLMDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAILNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1215..1234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1406..1785
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1786..1989
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1940..1960
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2031..2050
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2031..2049
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2081..2090
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2126..2146
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2189..2209
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4309..4326
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4349..4366)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4355..4579
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4650..4666)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4650..4666)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4663..4685)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4674..4690
                     /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(4698..4704,4705..4722,4723..4728))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4743..4764
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4881..4898)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5052..5640)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5132..5151)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacactcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttagta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccataa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc cgatccccta
     1201 gggtcgactc tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct
     1261 gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa
     1321 ggcttgaccg acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc
     1381 gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca
     1441 attacggggt cattagttca tagcccatat attgagttcc gcgttacata acttacggta
     1501 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     1561 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     1621 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     1681 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     1741 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     1801 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     1861 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     1921 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     1981 agcagagctg gtttagtgaa ccgtcagatc cgctagagat ccgcggccgc taatacgact
     2041 cactataggg agacccaagc ttggtaccga gctcggatcc gccaccatgg gcaaacggac
     2101 agccgacgga agcgagttcg agtcaccgaa aaagaagcga aaagttggct ccggcaaaag
     2161 aaccgccgac ggcagcgaat tcgagtcacc caagaagaag aggaaagtcg gctccggcca
     2221 cctgaaggag ctgctggaga tcaccgacac cacagagagg gatcgctccc tgcggagagc
     2281 cttctctccc tacaccgcca tgatcgacat cacaggctcc gaggccgtgg ccctgatcat
     2341 cctgctgaat ctgacatatc gcaagaacca ggtggacgat ctgctggaca agaagctggc
     2401 caagcaggcc ctgaagagcg aggatcacat caacaagtgc atcaaggaga tcgcctggtt
     2461 tcacacccac aatctgaagt acccagatat ccgggtgtcc aagcagaacc tggccgtgga
     2521 gccccctaca ctgcactctt atgtgctgag ctccgccaat taccccaagg cctatggctg
     2581 gagccacgac tccgccaagg tgaactttgc caagctgttc gtgagctact ttaagtggca
     2641 gaaccaggtg tcctggctgg cccaggtgct ggccaccaat agcgacaact ggaagtctgc
     2701 cttcacaagc ctgggcctgt ccgtgaaggc cttcaagagc ctgtgcgtga ccgtgaagaa
     2761 tagcctgcct gaggaggcca tcccagactc cgtggatagg tattctcgcc agatcagaat
     2821 gccataccac gacggctatc tggccgtgac ccccgtgatc agccacgtgg tgcagtccaa
     2881 gatccagcag gccgccatcg ataagcgggc cagattctcc aacgtggagt ttacacgccc
     2941 agcaaacgtg tctatgctgg ccgccagcct gggaggcgtg atcaatgtgc tgaactaccc
     3001 accctatatc agaagcaagt accacggcct gtctaatagc agggccttca agctgaacaa
     3061 tggccagacc gtgtttaacg tggaggccct gctgaagcct gagctgatca aggccctgga
     3121 gggcatcatc ttctccaaca atgccctggc cctgaagcag aggcgccagc agaaggtgaa
     3181 gaatatcaag gagctgcgca acacactgct ggagtggttc tctccagtgt ttgagtggcg
     3241 gctggacgcc atagagaatg gctacgatct ggagcagctg gagtccgcct ctgagagact
     3301 ggagtataag atcctgtccc tgcccgacaa tgagctgcct tctctgacca tcccactgtt
     3361 cagactgctg aacgagatgc tgggcggcgt gagcatgaca cagaggtacg cctttcaccc
     3421 caagctgatg tcccctctga agcgggccct gcagtggctg ctggtgaatc tgaccgacca
     3481 gaagcacgtg ctgatcgagg aggacgatga gcactacaga tatctgcacc tgtccggcat
     3541 cagggtgttt gatgcccagg ccctgtctaa cccatactgc tctggcatcc ccagcctgac
     3601 agccgtgtgg ggaatgatcc actcttatca gaggaagctg aatgaggccc tgggcaccaa
     3661 cgtgcggttc accagcttca gctggttcat cagaaactac agcgccgtgg caggcaagaa
     3721 gctgcccgag ctgagcctgc agggagcaca gcagtccaga ctgaagaggc ctggcatcat
     3781 cgacggcaag tactgtgacc tggtgttcga tctgatcatc cacatcgatg gctatgagga
     3841 cgatctgcag gccgtggaca gcaagcctga tatcctgaag gcccacttcc catccaattt
     3901 tgcaggaggc gtgatgcacc agccagagct gaactctaat atcaactggt gctgtctgta
     3961 tagcaatgag aaccagctgt ttgagaagct gcggagactg cccctgtccg gctgctgggt
     4021 catgcctacc gagcacaaga tccaggacct ggatgagctg ctgctgctgc tgaactctga
     4081 cagcaagctg tccccttcta tgatgggcta catgctgctg accgagccaa tggcccgcgt
     4141 gggcagcctg gagcggctgc actgttacgc cgagcctgcc atcggcgtgg tgaagtatga
     4201 ggccgccaca tctgtgaggc tgaagggcat cggcaattat ttcaacagcg ccttttggat
     4261 gctggatgcc caggagaagt tcatgctgat gaagaaggtg taaccggtca tcatcaccat
     4321 caccattgag tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     4381 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     4441 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     4501 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     4561 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc gataccgtcg
     4621 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4681 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctagggtgcc
     4741 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     4801 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4861 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4921 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4981 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5041 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5101 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     5161 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     5221 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     5281 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     5341 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     5401 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     5461 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     5521 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     5581 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
//
