LOCUS       sequence-393154-        4845 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a mini-transposon derived from Pseudoalteromonas Tn7016 with
            a SA-T2A-PuroR-T2A-eGFP cargo. Total transposon size = 2,024 bp..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4845)
  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 4845)
  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: pSL3936 (pDonor(AAVS1)_Pse)
FEATURES             Location/Qualifiers
     source          1..4845
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(125..144)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     244..266
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(304..322)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        390..494
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(495..563,564..1355)
                     /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(713..732)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1526..2114
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2015..2034
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2268..2285
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2402..2423
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2438..2443,2444..2461,2462..2468)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2476..2492
                     /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     2481..2503
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2500..2516
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2500..2516
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    2575..2696
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2586..2605)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2640..2659
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    2841..2866
                     /label=SA
                     /note="splice acceptor site"
     CDS             2890..2943
                     /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             2953..3549
                     /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(2953..2972)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3449..3469
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3559..3612
                     /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(3613..3615,3616..4326)
                     /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(3655..3676)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3916..3935)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4263..4284
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4354..4371)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4360..4567
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4745..4762)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4745..4761)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4754..4776)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc
       61 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
      121 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
      181 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
      241 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
      301 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
      361 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      421 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
      481 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
      541 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
      601 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
      661 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
      721 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
      781 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
      841 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
      901 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
      961 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1021 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1081 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1141 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1201 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1261 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     1321 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     1381 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     1441 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     1501 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     1561 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     1621 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     1681 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     1741 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     1801 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     1861 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     1921 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     1981 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2041 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2101 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2161 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2221 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     2281 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     2341 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     2401 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     2461 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     2521 attacgccaa cgtttaataa ataataaata ataaaataaa aaataaaaaa taaataagat
     2581 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
     2641 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaagc
     2701 tttgtcgctg caaccatagt ttgacataat gcaaccatac cttgacataa ttaaagtcat
     2761 actctgacat aaagcccgat cagcattagc aagacggctc acgagatgga gccagagagg
     2821 atcctggatc gtgtcttcga ctgacctctt ctcttcctcc cacagggcct cgagagatct
     2881 ggcagcggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc
     2941 cctaggctcg agatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     3001 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     3061 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     3121 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     3181 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     3241 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     3301 cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc
     3361 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     3421 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     3481 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     3541 cccggtgccg gcagtggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag
     3601 aatcctggcc cagtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     3661 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     3721 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     3781 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     3841 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     3901 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     3961 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     4021 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     4081 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     4141 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     4201 gacaaccact acctgagcac ccagtgcgcc ctgagcaaag accccaacga gaagcgcgat
     4261 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     4321 tacaaggaat tctaactaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     4381 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     4441 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     4501 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg
     4561 ctggggatcg aagacacagt cggctggttt ttttgttttt agcgttataa cttttatttc
     4621 actatgtcac tgtatggttg cattttatgt cagtgaatgg ttgcaggttt tttgtaagtt
     4681 attgatatgc ctatccagtg tatgtcacgc tttgcttgca gcgacaggta ccgagctcga
     4741 attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     4801 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcg
//
