LOCUS       sequence-475960-        4191 bp    DNA     circular SYN 10-JUL-2025
DEFINITION  Plasmid expressing mRuby2 and PuroR with the hPGK promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4191)
  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 4191)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 10, 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: pKG2439_pGEEC535_pShip-hPGK-mRuby2-P2A-PuroR-bGH
FEATURES             Location/Qualifiers
     source          1..4191
                     /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"
     promoter        2057..2567
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2486..2504
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      2573..2582
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2579..3289
                     /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             3299..3355
                     /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             3356..3955
                     /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(3356..3375)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3852..3872
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3967..4191
                     /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 cttactataa ggatccgggg ttggggttgc gccttttcca aggcagccct gggtttgcgc
     2101 agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctggatctc
     2161 gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc
     2221 ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt
     2281 gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc
     2341 agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag
     2401 ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag
     2461 tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt
     2521 cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagaaa tggccaccat
     2581 ggtgtctaag ggcgaagagc tgatcaagga aaatatgcgt atgaaggtgg tcatggaagg
     2641 ttcggtcaac ggccaccaat tcaaatgcac aggtgaagga gaaggcaatc cgtacatggg
     2701 aactcaaacc atgaggatca aagtcatcga gggaggaccc ctgccatttg cctttgacat
     2761 tcttgccacg tcgttcatgt atggcagccg tacttttatc aagtacccga aaggcattcc
     2821 tgatttcttt aaacagtcct ttcctgaggg ttttacttgg gaaagagtta cgagatacga
     2881 agatggtgga gtcgtcaccg tcatgcagga caccagcctt gaggatggct gtctcgttta
     2941 ccacgtccaa gtcagagggg taaactttcc ctccaatggt cccgtgatgc agaagaagac
     3001 caagggttgg gagcctaata cagagatgat gtatccagca gatggtggtc tgaggggata
     3061 cactcatatg gcactgaaag ttgatggtgg tggccatctg tcttgctctt tcgtaacaac
     3121 ttacaggtca aaaaagaccg tcgggaacat caagatgccc ggtatccatg ccgttgatca
     3181 ccgcctggaa aggttagagg aaagtgacaa tgaaatgttc gtagtacaac gcgaacacgc
     3241 agttgccaag ttcgccgggc ttggtggtgg gatggacgag ctgtacaagg gaagcggagc
     3301 tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg gacctatgac
     3361 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac
     3421 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca
     3481 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     3541 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     3601 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     3661 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     3721 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     3781 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     3841 atccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     3901 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacaacg
     3961 ttgcacctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     4021 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     4081 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     4141 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg g
//
