LOCUS       sequence-475959-        4881 bp    DNA     circular SYN 10-JUL-2025
DEFINITION  Plasmid expressing mRuby2 and PuroR with the UbC promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4881)
  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 4881)
  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: pKG2440_pGEEC536_pShip-UbC-mRuby2-P2A-PuroR-bGH
FEATURES             Location/Qualifiers
     source          1..4881
                     /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        2047..3258
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3095..3114
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     regulatory      3263..3272
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3269..3979
                     /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             3989..4045
                     /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             4046..4645
                     /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(4046..4065)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4542..4562
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4657..4881
                     /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 cttactggcc tccgcgccgg gttttggcgc ctcccgcggg cgcccccctc ctcacggcga
     2101 gcgctgccac gtcagacgaa gggcgcagcg agcgtcctga tccttccgcc cggacgctca
     2161 ggacagcggc ccgctgctca taagactcgg ccttagaacc ccagtatcag cagaaggaca
     2221 ttttaggacg ggacttgggt gactctaggg cactggtttt ctttccagag agcggaacag
     2281 gcgaggaaaa gtagtccctt ctcggcgatt ctgcggaggg atctccgtgg ggcggtgaac
     2341 gccgatgatt atataaggac gcgccgggtg tggcacagct agttccgtcg cagccgggat
     2401 ttgggtcgcg gttcttgttt gtggatcgct gtgatcgtca cttggtgagt agcgggctgc
     2461 tgggctggcc ggggctttcg tggccgccgg gccgctcggt gggacggaag cgtgtggaga
     2521 gatcgccaag ggctgtagtc tgggtccgcg agcaaggttg ccctgaactg ggggttgggg
     2581 ggagcgcagc aaaatggcgg ctgttcccga gtcttgaatg gaagacgctt gtgaggcggg
     2641 ctgtgaggtc gttgaaacaa ggtggggggc atggtgggcg gcaagaaccc aaggtcttga
     2701 ggccttcgct aatgcgggaa agctcttatt cgggtgagat gggctggggc accatctggg
     2761 gaccctgacg tgaagtttgt cactgactgg agaactcggt ttgtcgtctg ttgcgggggc
     2821 ggcagttatg gcggtgccgt tgggcagtgc acccgtacct ttgggagcgc gcgccctcgt
     2881 cgtgtcgtga cgtcacccgt tctgttggct tataatgcag ggtggggcca cctgtcggta
     2941 ggtgtgcggt aggcttttct ccgtcgcagg acgcagggtt cgggcctagg gtaggctctc
     3001 ctgaatcgac aggcgccgga cctctggtga ggggagggat aagtgaggcg tcagtttctt
     3061 tggtcggttt tatgtaccta tcttcttaag tagctgaagc tccggttttg aactatgcgc
     3121 tcggggttgg cgagtgtgtt ttgtgaagtt ttttaggcac cttttgaaat gtaatcattt
     3181 gggtcaatat gtaattttca gtgttagact agtaaattgt ccgctaaatt ctggccgttt
     3241 ttggcttttt tgttagacaa tggccaccat ggtgtctaag ggcgaagagc tgatcaagga
     3301 aaatatgcgt atgaaggtgg tcatggaagg ttcggtcaac ggccaccaat tcaaatgcac
     3361 aggtgaagga gaaggcaatc cgtacatggg aactcaaacc atgaggatca aagtcatcga
     3421 gggaggaccc ctgccatttg cctttgacat tcttgccacg tcgttcatgt atggcagccg
     3481 tacttttatc aagtacccga aaggcattcc tgatttcttt aaacagtcct ttcctgaggg
     3541 ttttacttgg gaaagagtta cgagatacga agatggtgga gtcgtcaccg tcatgcagga
     3601 caccagcctt gaggatggct gtctcgttta ccacgtccaa gtcagagggg taaactttcc
     3661 ctccaatggt cccgtgatgc agaagaagac caagggttgg gagcctaata cagagatgat
     3721 gtatccagca gatggtggtc tgaggggata cactcatatg gcactgaaag ttgatggtgg
     3781 tggccatctg tcttgctctt tcgtaacaac ttacaggtca aaaaagaccg tcgggaacat
     3841 caagatgccc ggtatccatg ccgttgatca ccgcctggaa aggttagagg aaagtgacaa
     3901 tgaaatgttc gtagtacaac gcgaacacgc agttgccaag ttcgccgggc ttggtggtgg
     3961 gatggacgag ctgtacaagg gaagcggagc tactaacttc agcctgctga agcaggctgg
     4021 agacgtggag gagaaccctg gacctatgac cgagtacaag cccacggtgc gcctcgccac
     4081 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     4141 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     4201 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     4261 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     4321 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     4381 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     4441 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     4501 gcgcgccggg gtgcccgcct tcctggagac atccgcgccc cgcaacctcc ccttctacga
     4561 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     4621 catgacccgc aagcccggtg cctgacaacg ttgcacctgt gccttctagt tgccagccat
     4681 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     4741 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     4801 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     4861 gggatgcggt gggctctatg g
//
