LOCUS       sequence-492544-        4132 bp    DNA     circular SYN 11-NOV-2025
DEFINITION  IVT template for making modRNA encoding PuroR-P2A-iCre.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4132)
  AUTHORS   Kabaria SR, Bae Y, Ehmann ME, Lende-Dorn BA, Beitz AM, Peterman EL, 
            Love KS, Ploessl DS, Galloway KE
  TITLE     Programmable promoter editing for precise control of transgene 
            expression.
  JOURNAL   Nat Biotechnol. 2025 Oct 13. doi: 10.1038/s41587-025-02854-y.
  PUBMED    41083901
REFERENCE   2  (bases 1 to 4132)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 11, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2025 Oct 13. doi: 10.1038/s41587-025-02854-y."
COMMENT     Sequence Label: pIVT-PuroR-P2A-iCre-polyA_pKG03717
FEATURES             Location/Qualifiers
     source          1..4132
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      180..768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     669..688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             897..1493
                     /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(897..916)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1393..1413
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1503..1559
                     /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             1566..1586
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    2717..3111
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     primer_bind     complement(3402..3421)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             join(3413..4132,1..90)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3480..3499)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
       61 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacattat
      121 tcagattggg ccccgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
      181 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
      241 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
      301 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
      361 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      421 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      481 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      541 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      601 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      661 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      721 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
      781 gcggcctttt tacggttcct ggcctaatac gactcactat aaggctcctg ggcaacgtgc
      841 tggtctgtgt gctggcccat cactttggca aagcacgcta ccggtcaatg gccaccatga
      901 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
      961 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     1021 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     1081 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     1141 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     1201 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     1261 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     1321 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     1381 catccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     1441 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gccggaagcg
     1501 gagctactaa cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggaccta
     1561 tggtgcccaa gaagaagagg aaagtctcca acctgctgac tgtgcaccaa aacctgcctg
     1621 ccctccctgt ggatgccacc tctgatgaag tcaggaagaa cctgatggac atgttcaggg
     1681 acaggcaggc cttctctgaa cacacctgga agatgctcct gtctgtgtgc agatcctggg
     1741 ctgcctggtg caagctgaac aacaggaaat ggttccctgc tgaacctgag gatgtgaggg
     1801 actacctcct gtacctgcaa gccagaggcc tggctgtgaa gaccatccaa cagcacctgg
     1861 gccagctcaa catgctgcac aggagatctg gcctgcctcg cccttctgac tccaatgctg
     1921 tgtccctggt gatgaggaga atcagaaagg agaatgtgga tgctggggag agagccaagc
     1981 aggccctggc ctttgaacgc actgactttg accaagtcag atccctgatg gagaactctg
     2041 acagatgcca ggacatcagg aacctggcct tcctgggcat tgcctacaac accctgctgc
     2101 gcattgccga aattgccaga atcagagtga aggacatctc ccgcaccgat ggtgggagaa
     2161 tgctgatcca cattggcagg accaagaccc tggtgtccac agctggtgtg gagaaggccc
     2221 tgtccctggg ggttaccaag ctggtggaga gatggatctc tgtgtctggt gtggctgatg
     2281 accccaacaa ctacctgttc tgccgggtca gaaagaatgg tgtggctgcc ccttctgcca
     2341 cctcccaact gtccacccgg gccctggaag ggatctttga ggccacccac cgcctgatct
     2401 atggtgccaa ggatgactct gggcagagat acctggcctg gtctggccac tctgccagag
     2461 tgggtgctgc cagggacatg gccagggctg gtgtgtccat ccctgaaatc atgcaggctg
     2521 gtggctggac caatgtgaac attgtgatga actacatcag aaacctggac tctgagactg
     2581 gggccatggt gaggctgctc gaggatgggg actgacaacc tcgagctcaa gcttcgaatt
     2641 caccccacca gtgcaggctg cctatcagaa agtggtggct ggtgtggcta atgccctggc
     2701 ccacaagtat cactaagctc gctttcttgc tgtccaattt ctattaaagg ttcctttgtt
     2761 ccctaagtcc aactactaaa ctgggggata ttatgaaggg ccttgagcat ctggattctg
     2821 cctaataaaa aacatttatt ttcattgcaa tgatgtattt aaattatttc tgaatatttt
     2881 actaaaaagg gaatgtggga ggtcagtgca tttaaaacat aaagaaatga agagctagtt
     2941 caaaccttgg gaaaatacac tatatcttaa actccatgaa agaaggtgag gctgcaaaca
     3001 gctaatgcac attggcaaca gcccctgatg cctatgcctt attcatccct cagaaaagga
     3061 ttcaagtaga ggcttgattt ggaggttaaa gttttgctat gctgtatttt accgaatcgg
     3121 attttgtttt taatatttca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3181 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3241 aaaaaaaaaa aaaaaaaaaa aaaaaacgcg tcgaagggaa ttaattcttg aagacgaaag
     3301 ggccaggtgg cacgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     3361 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     3421 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     3481 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     3541 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     3601 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     3661 gcattttatc cgtactcctg gtgatgcatg gttactcacc actgcgatcc ccggaaaaac
     3721 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     3781 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     3841 cgtatttcgt ctagctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     3901 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     3961 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     4021 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     4081 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt ca
//
