LOCUS       sequence-489522-        5008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IVT template for making modRNA encoding mRuby2-P2A-PuroR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5008)
  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 5008)
  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. 2025 Oct 13. doi: 10.1038/s41587-025-02854-y."
COMMENT     Sequence Label: pIVT-mRuby2-P2A-PuroR_pKG02755
FEATURES             Location/Qualifiers
     source          1..5008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      5..14
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             11..721
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             731..787
                     /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             788..1387
                     /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(788..807)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1284..1304
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     promoter        3222..3326
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3327..3395,3396..4187)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGYRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3545..3564)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4358..4946
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4847..4866
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aatggccacc atggtgtcta agggcgaaga gctgatcaag gaaaatatgc gtatgaaggt
       61 ggtcatggaa ggttcggtca acggccacca attcaaatgc acaggtgaag gagaaggcaa
      121 tccgtacatg ggaactcaaa ccatgaggat caaagtcatc gagggaggac ccctgccatt
      181 tgcctttgac attcttgcca cgtcgttcat gtatggcagc cgtactttta tcaagtaccc
      241 gaaaggcatt cctgatttct ttaaacagtc ctttcctgag ggttttactt gggaaagagt
      301 tacgagatac gaagatggtg gagtcgtcac cgtcatgcag gacaccagcc ttgaggatgg
      361 ctgtctcgtt taccacgtcc aagtcagagg ggtaaacttt ccctccaatg gtcccgtgat
      421 gcagaagaag accaagggtt gggagcctaa tacagagatg atgtatccag cagatggtgg
      481 tctgagggga tacactcata tggcactgaa agttgatggt ggtggccatc tgtcttgctc
      541 tttcgtaaca acttacaggt caaaaaagac cgtcgggaac atcaagatgc ccggtatcca
      601 tgccgttgat caccgcctgg aaaggttaga ggaaagtgac aatgaaatgt tcgtagtaca
      661 acgcgaacac gcagttgcca agttcgccgg gcttggtggt gggatggacg agctgtacaa
      721 gggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc
      781 tggacctatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
      841 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
      901 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
      961 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     1021 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     1081 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     1141 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     1201 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     1261 cttcctggag acatccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     1321 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     1381 tgcctgacaa cctcgagctc aagcttcgaa ttcaccccac cagtgcaggc tgcctatcag
     1441 aaagtggtgg ctggtgtggc taatgccctg gcccacaagt atcactaagc tcgctttctt
     1501 gctgtccaat ttctattaaa ggttcctttg ttccctaagt ccaactacta aactggggga
     1561 tattatgaag ggccttgagc atctggattc tgcctaataa aaaacattta ttttcattgc
     1621 aatgatgtat ttaaattatt tctgaatatt ttactaaaaa gggaatgtgg gaggtcagtg
     1681 catttaaaac ataaagaaat gaagagctag ttcaaacctt gggaaaatac actatatctt
     1741 aaactccatg aaagaaggtg aggctgcaaa cagctaatgc acattggcaa cagcccctga
     1801 tgcctatgcc ttattcatcc ctcagaaaag gattcaagta gaggcttgat ttggaggtta
     1861 aagtctgaga gatcccctca taatttcccc aaagcgtaac catgtgtgaa taaattttga
     1921 gctagtaggg ttgcagccac gagtaagtct tcccttgtta ttgtgtagcc agaatgccgc
     1981 aaaacttcca tgcctaagcg aactgttgag agtacgtttc gatttctgac tgtgttagcc
     2041 tggaagtgct tgtcccaacc ttgtttctga gcatgaacgc ccgcaagcca acatgttagt
     2101 tgaagcatca gggcgattag cagcatgata tcaaaacgct ctgagctgct cgttcggcta
     2161 tggcgtaggc ctagtccgta ggcaggactt ttcaagtctc ggaaggtttc ttcaatctgc
     2221 attcgcttcg aatagatatt aacaagttgt ttgggtgttc gaatttcaac aggtaagtta
     2281 gttgctagaa cccatggctc ctttgccgac gctgagtaga ttttaggtga cgggtggtga
     2341 caatgagtcc gtgtcgagcg ctgatttttt cggcctttag agcgagattt atacaataga
     2401 atttggcatg agattggatt gcttttagtc agcctcttat agcctaaagt ctttgagtga
     2461 ctagatgaca tatcatgtaa gttgctgata ggtttccagt tttccgctcc taggtctgca
     2521 tattgtactt ttcctcttac tcgacttaac cagtaccaac ccagcttctc aacggattta
     2581 taccatggca ctttaaagcc agcatcactg acaatgagcg gtgtggtgtt actcggtaga
     2641 atgctcgcaa ggtcggctag aaattggtca tgagctttct ttgaacattg ctctgaaagc
     2701 gggaacgctt tctcataaag agtaacagaa cgaccgtgta gtgcgactga agctcgcaat
     2761 accataagtc gtttttgctc acgaatatca gaccagtcaa caagtacaat gggcatcgta
     2821 ttgcccgaac agataaagct agcatgccaa cggtatacag cgagtcgctc tttgtggagg
     2881 tgacgattac ctaacaatcg gtcgattcgt ttgatgttat gttttgttct cgctttggtt
     2941 ggcaggttac ggccaagttc ggtaagagtg agagttttac agtcaagtaa tgcgtggcaa
     3001 gccaacgtta agctgttgag tcgttttaag tgtaattcgg ggcagaattg gtaaagagag
     3061 tcgtgtaaaa tatcgagttc gcacatcttg ttgtctgatt attgattttt cgcgaaacca
     3121 tttgatcata tgacaagatg tgtatccacc ttaacttaat gatttttacc aaaatcatta
     3181 ggggattcat cagggttaaa gttttgctat gctgtatttt acgcggaacc cctatttgtt
     3241 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3301 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3361 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3421 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3481 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3541 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     3601 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3661 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3721 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3781 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3841 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3901 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3961 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     4021 aatctggagc cggtgagcgt gggtatcgcg gtatcattgc agcactgggg ccagatggta
     4081 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4141 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4201 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4261 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4321 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4381 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4441 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4501 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4561 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4621 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4681 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4741 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4801 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4861 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4921 cgtcaggggg gcggagccta tggaaactcc tgggcaacgt gctggtctgt gtgctggccc
     4981 atcactttgg caaagcacgc taccggtc
//
