LOCUS       sequence-475970-        3494 bp    DNA     circular SYN 10-JUL-2025
DEFINITION  Plasmid expressing mRuby2 from the hPGK canonical promoter with the 
            CMV 5' UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3494)
  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 3494)
  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: 
            pKG3775_pGEEC582_pShip-hPGK(promoter)-CMV(UTR)-mRuby2-bGH
FEATURES             Location/Qualifiers
     source          1..3494
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      18..45
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     53..76
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     primer_bind     552..570
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      605..614
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             611..1321
                     /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"
     polyA_signal    1336..1560
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1671..1690)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1682..2491
                     /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(1749..1768)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2581..3169
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3070..3089
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      3334..3420
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 caggcatcaa actaagcaga aggccatcct gacggatggc ctttttgcgt ttctacaaac
       61 tcttcctgtt agttagttac ttaagctcgg gcccacctgc agtctactta ctataaggat
      121 ccggggttgg ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc
      181 tctgggcgtg gttccgggaa acgcagcggc gccgaccctg gatctcgcac attcttcacg
      241 tccgttcgca gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt
      301 cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa
      361 acggaagccg cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca
      421 gcgcgccgac cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc
      481 agcggccggg aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc
      541 ctgcccgcgc ggtgttccgc attctgcaag cagatcgcct ggagacgcca tccacgctgt
      601 aatggccacc atggtgtcta agggcgaaga gctgatcaag gaaaatatgc gtatgaaggt
      661 ggtcatggaa ggttcggtca acggccacca attcaaatgc acaggtgaag gagaaggcaa
      721 tccgtacatg ggaactcaaa ccatgaggat caaagtcatc gagggaggac ccctgccatt
      781 tgcctttgac attcttgcca cgtcgttcat gtatggcagc cgtactttta tcaagtaccc
      841 gaaaggcatt cctgatttct ttaaacagtc ctttcctgag ggttttactt gggaaagagt
      901 tacgagatac gaagatggtg gagtcgtcac cgtcatgcag gacaccagcc ttgaggatgg
      961 ctgtctcgtt taccacgtcc aagtcagagg ggtaaacttt ccctccaatg gtcccgtgat
     1021 gcagaagaag accaagggtt gggagcctaa tacagagatg atgtatccag cagatggtgg
     1081 tctgagggga tacactcata tggcactgaa agttgatggt ggtggccatc tgtcttgctc
     1141 tttcgtaaca acttacaggt caaaaaagac cgtcgggaac atcaagatgc ccggtatcca
     1201 tgccgttgat caccgcctgg aaaggttaga ggaaagtgac aatgaaatgt tcgtagtaca
     1261 acgcgaacac gcagttgcca agttcgccgg gcttggtggt gggatggacg agctgtacaa
     1321 gtaacaacgt tgcacctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     1381 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     1441 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     1501 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     1561 cgctcgctga ctgcaggtgc ccgtgtctca aaatctctga tgttacattg cacaagataa
     1621 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     1681 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
     1741 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     1801 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     1861 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     1921 gaccatcaag cattttatcc gtactcctgg tgatgcatgg ttactcacca ctgcgatccc
     1981 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     2041 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     2101 cagcgatcgc gtatttcgtc tagctcaggc gcaatcacga atgaataacg gtttggttga
     2161 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     2221 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     2281 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     2341 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     2401 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     2461 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacatta
     2521 ttcagattgg gccccgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     2581 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     2641 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     2701 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     2761 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     2821 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     2881 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     2941 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3001 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3061 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3121 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3181 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3241 gttatcccct gattctgtgg ataaccgtat taccgctagc atggatctcg gggacgtcta
     3301 actactaagc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcggaa
     3361 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     3421 ccgccgggag cggatttgaa cgttgtgaag caacggcccg gagggtggcg ggcaggacgc
     3481 ccgccataaa ctgc
//
