LOCUS       sequence-393155-        5234 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a mini-transposon derived from Pseudoalteromonas Tn7016 with
            a splice acceptor-T2A-PuroR-T2A-eGFP cargo, and a CRISPR RNA under a
            U6 promoter. Total transposon size = 2,024 bp..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5234)
  AUTHORS   Lampe GD, King RT, Halpin-Healy TS, Klompe SE, Hogan MI, Vo PLH, 
            Tang S, Chavez A, Sternberg SH
  TITLE     Targeted DNA integration in human cells without double-strand breaks
            using CRISPR-associated transposases.
  JOURNAL   Nat Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1.
  PUBMED    36991112
REFERENCE   2  (bases 1 to 5234)
  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: "Nat 
            Biotechnol. 2023 Mar 29. doi: 10.1038/s41587-023-01748-1."
COMMENT     Sequence Label: pSL5069 (pDonor(AAVS1),CRISPR_Pse)
FEATURES             Location/Qualifiers
     source          1..5234
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      774..1362
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1263..1282
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1516..1533
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1537..1785
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1537..1557
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1708..1727
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     1787..1806
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1787..1805
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1824..1838
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     protein_bind    2039..2060
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2075..2080,2081..2098,2099..2105)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2113..2129
                     /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)."
     primer_bind     2118..2140
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2137..2153
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2137..2153
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2212..2333
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2223..2242)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2277..2296
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    2478..2503
                     /label=SA
                     /note="splice acceptor site"
     CDS             2527..2580
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2590..3186
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2590..2609)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3086..3106
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3196..3249
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(3250..3252,3253..3963)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQCALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3292..3313)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3553..3572)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3900..3921
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3991..4008)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3997..4204
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4382..4399)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4382..4398)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4391..4413)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4607..4626)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4726..4748
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4786..4804)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4872..4976
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4977..5045,5046..5234,1..603)
                     /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     complement(5195..5214)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
       61 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
      121 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
      181 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
      241 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
      301 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
      361 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
      421 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
      481 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
      541 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
      601 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
      661 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
      721 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
      781 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
      841 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
      901 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
      961 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     1021 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     1081 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     1141 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     1201 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     1261 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     1321 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     1381 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     1441 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     1501 cgaacgaccg agcgcagcga gtcagtgagc gaggacgagg gcctatttcc catgattcct
     1561 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     1621 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1681 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1741 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgtaat acgactcact
     1801 atagggtcta gaattcctgt gacctgccgt ataggcagct gaagatacag tggggccact
     1861 agggacagga ttggtgacat aattctgccg aaaaggcagt gagtagtgta gcggccgctt
     1921 tttttttgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     1981 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     2041 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     2101 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     2161 acgccaacgt ttaataaata ataaataata aaataaaaaa taaaaaataa ataagataca
     2221 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     2281 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaagcttt
     2341 gtcgctgcaa ccatagtttg acataatgca accatacctt gacataatta aagtcatact
     2401 ctgacataaa gcccgatcag cattagcaag acggctcacg agatggagcc agagaggatc
     2461 ctggatcgtg tcttcgactg acctcttctc ttcctcccac agggcctcga gagatctggc
     2521 agcggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccct
     2581 aggctcgaga tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     2641 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     2701 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     2761 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     2821 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     2881 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     2941 cccaaggagc ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag
     3001 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     3061 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     3121 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     3181 ggtgccggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat
     3241 cctggcccag tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     3301 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     3361 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     3421 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     3481 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     3541 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     3601 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     3661 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     3721 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     3781 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     3841 aaccactacc tgagcaccca gtgcgccctg agcaaagacc ccaacgagaa gcgcgatcac
     3901 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     3961 aaggaattct aactagagct cgctgatcag cctcgactgt gccttctagt tgccagccat
     4021 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     4081 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     4141 ggggtggggt ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg
     4201 gggatcgaag acacagtcgg ctggtttttt tgtttttagc gttataactt ttatttcact
     4261 atgtcactgt atggttgcat tttatgtcag tgaatggttg caggtttttt gtaagttatt
     4321 gatatgccta tccagtgtat gtcacgcttt gcttgcagcg acaggtaccg agctcgaatt
     4381 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     4441 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     4501 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc
     4561 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
     4621 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4681 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4741 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     4801 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     4861 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     4921 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     4981 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5041 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5101 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5161 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5221 ttgacgccgg gcaa
//
