LOCUS       sequence-466030-        4782 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Engineered human-codon optimized PseTniQ in mammalian cells (for 
            evoCAST).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4782)
  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 4782)
  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-TniQ
FEATURES             Location/Qualifiers
     source          1..4782
                     /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             3451..3468
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3491..3508)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3497..3721
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3792..3808)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3792..3808)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3805..3827)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3816..3832
                     /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(3840..3846,3847..3864,3865..3870))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3885..3906
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4023..4040)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4194..4782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4274..4293)
                     /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 gctccggcgc
     2221 cttcctgttt tctcccaagg cccgggcctt tagcgacgag tccctggagt cttatctgct
     2281 gagagtggtg agcgagaatt tctttgatag ctacgagggc ctgtccctgg ccatcaggga
     2341 ggagctgcac gagctggact ttgaggcaca cggagcattc ccagtggacc tgaagcggct
     2401 gaacgtgtac cacgccaagc acaactctca cttcagaatg agagccctgg gactgctgga
     2461 gaccctgctg gacctgcccc ggtatgagct gcagaagctg gccctgctga agagcgatat
     2521 caagtttaac agctccgtgg ccctgtacaa caatggcgtg gacatccctc tgaggttcat
     2581 caggcaccac gcagaggagg cagtggattc tatcccagtg tgcagccagt gtctggccga
     2641 ggaggcctac atcaagcagt cctggcacat caagtgggtg aatgcctgca ccaagcacca
     2701 gtgtgccctg ctgcacaact gccccgagtg ttacgcccct atcaactata tcgagaatga
     2761 gtccatcaca cactgctctt gtggctttga gctgagctgc gcctctacca gccccgtgaa
     2821 tacactgtcc atcgagcacc tgaacaagct gctggacaag ggcgagagga acgattccaa
     2881 tcctctgttt aacaatatga ccctgacaga gaggttcgcc gccctgctgt ggtatcagga
     2941 gcggtactct cagaccgaca acttctgcct gaatgatgcc gtgaactact ttagcaagtg
     3001 gcctgccgtg ttcaatacag agctggacga gctgtccaag aacgccgaga tgaagctgat
     3061 cgatctgttt aacaagaccg agttcaagtt tatcttcggc gacgccatcc tggcctgtcc
     3121 aagcacacag aagcagtccg agtctcactt catctacagg gccctgctgg actatctggt
     3181 gaccctggtg gagtccaatc caaagacaaa gaagcccaat gccgccgatc tgctggtgtc
     3241 tgtgctggag gcagccaccc tgctgggcac aagcgtggag caggtgtacc gcctgtatca
     3301 gaatggcatc ctgcagaccg cctttcggca caagatgaat cagagaatca acccttacaa
     3361 gggcgccttc tttctgaggc acgtgatcga gtataagaca tccttcggca acgataaggc
     3421 ccgcatgtac ctgtctgcct ggtaaccggt catcatcacc atcaccattg agtttaaacc
     3481 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3541 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3601 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3661 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3721 gcttctgagg cggaaagaac cagctggggc tcgataccgt cgacctctag ctagagcttg
     3781 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     3841 aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc
     3901 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3961 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4021 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4081 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4141 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4201 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4261 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4321 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4381 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4441 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4501 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4561 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4621 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4681 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4741 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aa
//
