LOCUS       sequence-478038-        5394 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid expressing mRuby2 and PuroR with the CAG promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5394)
  AUTHORS   Peterman EL, Ploessl DS, Love KS, Sanabria V, Daniels RF, Johnstone 
            CP, Godavarti DR, Kabaria SR, Oakes CG, Pai AA, Galloway KE
  TITLE     High-resolution profiling reveals coupled transcriptional and 
            translational regulation of transgenes.
  JOURNAL   Nucleic Acids Research, Volume 53, Issue 11, 24 June 2025, gkaf528
  PUBMED    39651241
REFERENCE   2  (bases 1 to 5394)
  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: "Nucleic 
            Acids Research, Volume 53, Issue 11, 24 June 2025, gkaf528"
COMMENT     Sequence Label: pKG2441_pGEEC537_pShip-CAG-mRuby2-P2A-PuroR-bGH
FEATURES             Location/Qualifiers
     source          1..5394
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(111..130)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             122..931
                     /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(189..208)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1021..1609
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1510..1529
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      1774..1860
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1952..1979
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     1987..2010
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     enhancer        2109..2412
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          2707..3723
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3647..3669
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3731..3750
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      3776..3785
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3782..4492
                     /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             4502..4558
                     /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             4559..5158
                     /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(4559..4578)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5055..5075
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5170..5394
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 cgctcgctga ctgcaggtgc ccgtgtctca aaatctctga tgttacattg cacaagataa
       61 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
      121 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
      181 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
      241 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
      301 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
      361 gaccatcaag cattttatcc gtactcctgg tgatgcatgg ttactcacca ctgcgatccc
      421 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
      481 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
      541 cagcgatcgc gtatttcgtc tagctcaggc gcaatcacga atgaataacg gtttggttga
      601 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
      661 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
      721 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
      781 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
      841 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
      901 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacatta
      961 ttcagattgg gccccgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     1021 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     1081 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     1141 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     1201 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1261 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1321 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1381 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     1441 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     1501 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     1561 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     1621 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     1681 gttatcccct gattctgtgg ataaccgtat taccgctagc atggatctcg gggacgtcta
     1741 actactaagc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcggaa
     1801 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     1861 ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg ggcaggacgc
     1921 ccgccataaa ctgccaggca tcaaactaag cagaaggcca tcctgacgga tggccttttt
     1981 gcgtttctac aaactcttcc tgttagttag ttacttaagc tcgggcccac ctgcagtcta
     2041 cttactacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     2101 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     2161 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     2221 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     2281 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     2341 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     2401 gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc
     2461 tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg
     2521 gggggggggg gggggggggg gggggggggg ggggggcggg gggggggggg ggggggcgag
     2581 gggggggggg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
     2641 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
     2701 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
     2761 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
     2821 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
     2881 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt
     2941 gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt
     3001 gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg
     3061 gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg
     3121 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc
     3181 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg
     3241 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc
     3301 ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc
     3361 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag
     3421 agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc
     3481 cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
     3541 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc
     3601 tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     3661 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     3721 cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa aaatggccac
     3781 catggtgtct aagggcgaag agctgatcaa ggaaaatatg cgtatgaagg tggtcatgga
     3841 aggttcggtc aacggccacc aattcaaatg cacaggtgaa ggagaaggca atccgtacat
     3901 gggaactcaa accatgagga tcaaagtcat cgagggagga cccctgccat ttgcctttga
     3961 cattcttgcc acgtcgttca tgtatggcag ccgtactttt atcaagtacc cgaaaggcat
     4021 tcctgatttc tttaaacagt cctttcctga gggttttact tgggaaagag ttacgagata
     4081 cgaagatggt ggagtcgtca ccgtcatgca ggacaccagc cttgaggatg gctgtctcgt
     4141 ttaccacgtc caagtcagag gggtaaactt tccctccaat ggtcccgtga tgcagaagaa
     4201 gaccaagggt tgggagccta atacagagat gatgtatcca gcagatggtg gtctgagggg
     4261 atacactcat atggcactga aagttgatgg tggtggccat ctgtcttgct ctttcgtaac
     4321 aacttacagg tcaaaaaaga ccgtcgggaa catcaagatg cccggtatcc atgccgttga
     4381 tcaccgcctg gaaaggttag aggaaagtga caatgaaatg ttcgtagtac aacgcgaaca
     4441 cgcagttgcc aagttcgccg ggcttggtgg tgggatggac gagctgtaca agggaagcgg
     4501 agctactaac ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctat
     4561 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     4621 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     4681 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     4741 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     4801 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     4861 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     4921 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     4981 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     5041 gacatccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     5101 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaca
     5161 acgttgcacc tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     5221 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     5281 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     5341 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atgg
//
