LOCUS       sequence-475949-        3491 bp    DNA     circular SYN 10-JUL-2025
DEFINITION  Plasmid expressing mRuby2 from the hPGK canonical promoter with the 
            EFS 5' UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3491)
  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 3491)
  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: 
            pKG3776_pGEEC583_pShip-hPGK(promoter)-EFS(UTR)-mRuby2-bGH
FEATURES             Location/Qualifiers
     source          1..3491
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2..29
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     37..60
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     primer_bind     536..554
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      586..595
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             592..1302
                     /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    1317..1541
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1652..1671)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1663..2472
                     /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(1730..1749)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2562..3150
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3051..3070
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      3315..3401
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactcttcc tgttagttag
       61 ttacttaagc tcgggcccac ctgcagtcta cttactataa ggatccgggg ttggggttgc
      121 gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg cgtggttccg
      181 ggaaacgcag cggcgccgac cctggatctc gcacattctt cacgtccgtt cgcagcgtca
      241 cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa
      301 gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc
      361 tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat
      421 gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc cgggaagggg
      481 cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt
      541 ccgcattctg caagccaacg ggtttgccgc cagaacacag taatggccac catggtgtct
      601 aagggcgaag agctgatcaa ggaaaatatg cgtatgaagg tggtcatgga aggttcggtc
      661 aacggccacc aattcaaatg cacaggtgaa ggagaaggca atccgtacat gggaactcaa
      721 accatgagga tcaaagtcat cgagggagga cccctgccat ttgcctttga cattcttgcc
      781 acgtcgttca tgtatggcag ccgtactttt atcaagtacc cgaaaggcat tcctgatttc
      841 tttaaacagt cctttcctga gggttttact tgggaaagag ttacgagata cgaagatggt
      901 ggagtcgtca ccgtcatgca ggacaccagc cttgaggatg gctgtctcgt ttaccacgtc
      961 caagtcagag gggtaaactt tccctccaat ggtcccgtga tgcagaagaa gaccaagggt
     1021 tgggagccta atacagagat gatgtatcca gcagatggtg gtctgagggg atacactcat
     1081 atggcactga aagttgatgg tggtggccat ctgtcttgct ctttcgtaac aacttacagg
     1141 tcaaaaaaga ccgtcgggaa catcaagatg cccggtatcc atgccgttga tcaccgcctg
     1201 gaaaggttag aggaaagtga caatgaaatg ttcgtagtac aacgcgaaca cgcagttgcc
     1261 aagttcgccg ggcttggtgg tgggatggac gagctgtaca agtaacaacg ttgcacctgt
     1321 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1381 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1441 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     1501 agacaatagc aggcatgctg gggatgcggt gggctctatg gcgctcgctg actgcaggtg
     1561 cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgaaca
     1621 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg
     1681 gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg
     1741 gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg gaagcccgat
     1801 gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag
     1861 atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc
     1921 cgtactcctg gtgatgcatg gttactcacc actgcgatcc ccggaaaaac agcattccag
     1981 gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
     2041 cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt
     2101 ctagctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac
     2161 gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact tttgccattc
     2221 tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag
     2281 gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat
     2341 cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt
     2401 caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat
     2461 gagtttttct aatcagaatt ggttaattgg ttgtaacatt attcagattg ggccccgttc
     2521 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2581 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2641 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2701 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2761 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2821 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2881 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2941 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3001 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3061 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3121 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3181 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     3241 gataaccgta ttaccgctag catggatctc ggggacgtct aactactaag cgagagtagg
     3301 gaactgccag gcatcaaata aaacgaaagg ctcagtcgga agactgggcc tttcgtttta
     3361 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
     3421 acgttgtgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc
     3481 atcaaactaa g
//
