LOCUS       sequence-475973-        3246 bp    DNA     circular SYN 10-JUL-2025
DEFINITION  Plasmid expressing mRuby from an EF1a promoter lacking an intron, 
            used for modRNA production.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3246)
  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 3246)
  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: pKG3774_pGEEC580_pShip-EF1a(no_intron)-mRuby2-bGH
FEATURES             Location/Qualifiers
     source          1..3246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      10..37
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     45..68
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     promoter        123..334
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      349..358
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             355..1065
                     /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    1080..1304
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1415..1434)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1426..2235
                     /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(1493..1512)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2325..2913
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2814..2833
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      3078..3164
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aaactaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttcctg
       61 ttagttagtt acttaagctc gggcccacct gcagtctact tactgctccg gtgcccgtca
      121 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
      181 aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
      241 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
      301 tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgagaatggc caccatggtg
      361 tctaagggcg aagagctgat caaggaaaat atgcgtatga aggtggtcat ggaaggttcg
      421 gtcaacggcc accaattcaa atgcacaggt gaaggagaag gcaatccgta catgggaact
      481 caaaccatga ggatcaaagt catcgaggga ggacccctgc catttgcctt tgacattctt
      541 gccacgtcgt tcatgtatgg cagccgtact tttatcaagt acccgaaagg cattcctgat
      601 ttctttaaac agtcctttcc tgagggtttt acttgggaaa gagttacgag atacgaagat
      661 ggtggagtcg tcaccgtcat gcaggacacc agccttgagg atggctgtct cgtttaccac
      721 gtccaagtca gaggggtaaa ctttccctcc aatggtcccg tgatgcagaa gaagaccaag
      781 ggttgggagc ctaatacaga gatgatgtat ccagcagatg gtggtctgag gggatacact
      841 catatggcac tgaaagttga tggtggtggc catctgtctt gctctttcgt aacaacttac
      901 aggtcaaaaa agaccgtcgg gaacatcaag atgcccggta tccatgccgt tgatcaccgc
      961 ctggaaaggt tagaggaaag tgacaatgaa atgttcgtag tacaacgcga acacgcagtt
     1021 gccaagttcg ccgggcttgg tggtgggatg gacgagctgt acaagtaaca acgttgcacc
     1081 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     1141 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     1201 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     1261 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcgctcg ctgactgcag
     1321 gtgcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga
     1381 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     1441 cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     1501 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc
     1561 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     1621 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     1681 atccgtactc ctggtgatgc atggttactc accactgcga tccccggaaa aacagcattc
     1741 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     1801 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     1861 cgtctagctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     1921 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     1981 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     2041 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     2101 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     2161 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     2221 gatgagtttt tctaatcaga attggttaat tggttgtaac attattcaga ttgggccccg
     2281 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     2341 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     2401 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     2461 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     2521 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     2581 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     2641 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     2701 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     2761 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     2821 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     2881 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     2941 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     3001 gtggataacc gtattaccgc tagcatggat ctcggggacg tctaactact aagcgagagt
     3061 agggaactgc caggcatcaa ataaaacgaa aggctcagtc ggaagactgg gcctttcgtt
     3121 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     3181 tgaacgttgt gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     3241 ggcatc
//
