LOCUS       sequence-412776-        7455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of TnpBmax in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7455)
  AUTHORS   Marquart KF, Mathis N, Mollaysa A, Muller S, Kissling L, Rothgangl 
            T, Schmidheini L, Kulcsar PI, Allam A, Kaufmann MM, Matsushita M, 
            Haenggi T, Cathomen T, Kopf M, Krauthammer M, Schwank G
  TITLE     Effective genome editing with an enhanced ISDra2 TnpB system and 
            deep learning-predicted omegaRNAs.
  JOURNAL   Nat Methods. 2024 Sep 23. doi: 10.1038/s41592-024-02418-z.
  PUBMED    39313558
REFERENCE   2  (bases 1 to 7455)
  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 
            Methods. 2024 Sep 23. doi: 10.1038/s41592-024-02418-z."
COMMENT     Sequence Label: pKM-T4-CMV-TnpB-optimized-P2A-GFP-Puro
FEATURES             Location/Qualifiers
     source          1..7455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          676..808
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        820..838
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2157..2177
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2190..2255
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2274..2330
                     /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             join(2331..2333,2334..2336,2337..3047)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2331..2333,2334..2336,2337..3047)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2376..2397)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2637..2656)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2984..3005
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3078..3674
                     /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(3078..3097)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3574..3594
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3804..3925
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3841..3860)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3895..3914
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3942..3958)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3943..3959)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    3964..4012
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4348..4368)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4348..4366)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4387..4403)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4387..4403)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4400..4422)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4411..4427
                     /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(4435..4441,4442..4459,4460..4465))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4480..4501
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4618..4635)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4789..5377)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4869..4888)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5548..6339,6340..6408))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAAKLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6171..6190
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6409..6513)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6540..6995
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6627..6646)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6837..6858
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7121..7143
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7135..7152
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7136..7152
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        7162..7180
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag aggtcgttta gtgaaccgtc
      601 agatcactag tagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg
      661 ctcgactgat cacaggtaag tatcaaggtt acaagacagg tttaaggagg ccaatagaaa
      721 ctgggcttgt cgagacagag aagattcttg cgtttctgat aggcacctat tggtcttact
      781 gacatccact ttgcctttct ctccacaggg cgcggccgct aatacgactc actataggga
      841 gagccgccac catgatccgg aacaaggcct tcgtggtgcg cctgtaccct aatgccgctc
      901 agaccgagct gatcaacaga accctcggca gcgcccggtt tgtgtacaac cacttcctgg
      961 caagaagaat cgccgcttac aaggaatctg gcaagggcct gacctacggc caaacaagca
     1021 gcgagctgac cctgctgaag caggccgagg aaaccagctg gctgtctgag gtggacaagt
     1081 tcgctctgca gaatagcctg aagaacctgg aaaccgccta taagaatttc ttcagaaccg
     1141 tgaagcagag cggcaagaaa gtgggcttcc caagattcag aaagaagagg acaggcgaga
     1201 gctacagaac acagttcacc aacaacaaca tccaaatcgg cgagggcaga ctcaagctgc
     1261 ccaagctggg ctgggtcaag accaagggcc aacaggacat ccagggcaaa atcctgaacg
     1321 tgaccgtgcg gagaatccac gaaggccact acgaggcctc tgtgctgtgt gaagtggaaa
     1381 tcccctacct gcctgctgcc cctaaattcg ccgctggtgt tgacgtggga attaaggact
     1441 tcgccatcgt gaccgacggc gtgcggttta agcacgagca gaaccccaaa tactaccgga
     1501 gcaccctgaa aagactgcgg aaagcccagc agaccctgag cagacggaaa aaaggctccg
     1561 ccagatacgg aaaggccaag accaagctgg ctaggatcca taagcggatc gtgaacaaga
     1621 gacaggattt tctgcacaaa ctgacgacca gcctggtgag agagtacgag atcattggaa
     1681 cagagcacct gaagcctgat aacatgagaa agaaccgcag actggccctg agcatcagcg
     1741 atgccggctg gggagaattc atcagacagc tggaatacaa ggctgcctgg tacggcagac
     1801 ttgtttccaa ggtgtcccct tacttcccat ctagccagct gtgccacgat tgcggcttca
     1861 agaatcctga ggtgaagaac ctggccgtgc ggacatggac ctgtcctaac tgcggcgaga
     1921 cacacgaccg ggacgagaac gccgctctga acatcagacg ggaagccctg gtggccgccg
     1981 gcatcagcga cacactgaac gcccacggcg gatatgtgcg gcctgccagc gccggaaatg
     2041 gcctgagatc tgagaaccac gccacactgg tggtctccag agccgacccc aagaagaaga
     2101 ggaaaggcgg cggaggcgga agcgggaaaa gaaccgccga cggcagcgaa ttcgagccca
     2161 agaagaagag gaaagtctcg agcggaggcg actacaaaga ccatgacggt gattataaag
     2221 atcatgacat cgattacaag gatgacgatg acaagggagg cggtggaagc ggcgcaacaa
     2281 acttctctct gctgaaacaa gccggagatg tcgaagagaa tcctggaccg atggtgagca
     2341 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2401 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2461 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2521 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     2581 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2641 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2701 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2761 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     2821 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     2881 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2941 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     3001 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc ggactcagat
     3061 ctaggagacg accttccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     3121 acgtcccccg ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     3181 acaccgtcga cccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     3241 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     3301 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     3361 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     3421 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     3481 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     3541 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     3601 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     3661 gcaagcccgg tgcgggatcc accggatcta gataactgat cataatcagc cataccacat
     3721 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     3781 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     3841 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3901 tgtccaaact catcaatgta tcttaacgcg atcaagctta tcgataccgt cgacctcgag
     3961 ggcaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgtaattcga
     4021 gcatcttacc gccatttatt cccagatctt taaacaattt aaaggcaatg ctaccaaata
     4081 ctaagcgcgt gtatgtacac ttctgaccca ctgggaatgt gatgaaagaa ataaaagctg
     4141 aaatgaatca ttctctctac tattattctg atatttcaca ttcttaaaat aaagtggtga
     4201 tcctaactga ccttaagaca gggaatcttt actcggatta aatgtcagga attgtgaaaa
     4261 agtgagttta aatgtatttg gctaaggtgt atgtaaactt ccgacttcaa ctgtagttta
     4321 aacgggcccg gtacccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     4381 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4441 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4501 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4561 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4621 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4681 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4741 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4801 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4861 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4921 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaaccg tggcgctttc
     4981 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5041 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5101 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5161 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5221 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5281 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5341 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5401 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5461 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5521 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5581 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5641 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5701 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5761 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5821 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5881 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5941 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6001 aagtaacttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6061 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6121 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6181 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6241 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6301 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6361 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6421 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6481 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     6541 cgcgccctgt agcggcgcat taagcgcggc gggagtggtg gttacgcgca gcgtgaccgc
     6601 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     6661 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     6721 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     6781 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     6841 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     6901 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     6961 cgcgaatttt aacaaaatat taacgcttac aatttccatt cgccattcag gctgcgcaac
     7021 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     7081 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     7141 acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattgga gctagcgttt
     7201 aaacctacag ttgaagtcgg aagtttacat acacttaagt tggagtcatt aaaactcgtt
     7261 tttcaactac tccacaaatt tcttgttaac aaacaatagt tttggcaagt cagttaggac
     7321 atctactttg tgcatgacac aagtcatttt tccaacaatt gtttacagac agattatttc
     7381 acttataatt cactgtatca caattccagt gggtcagaag tgtacataca cgcgcttgac
     7441 tgtgccttta agctt
//
