LOCUS       sequence-475944-        3176 bp    DNA     circular SYN 10-JUL-2025
DEFINITION  BsaI Golden Gate assembly plasmid encoding the mRuby2-P2A-PuroR CDS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3176)
  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 3176)
  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: pKG2387_pPV2-mRuby2-P2A-PuroR
FEATURES             Location/Qualifiers
     source          1..3176
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      38..626
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     527..546
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     regulatory      651..660
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             657..1367
                     /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             1377..1433
                     /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             1434..2033
                     /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(1434..1453)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1930..1950
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     promoter        2078..2182
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2183..2251,2252..3043)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGYRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2401..2420)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
       61 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
      121 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
      181 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
      241 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
      301 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
      361 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
      421 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
      481 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
      541 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
      601 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgg gtctcaaatg gccaccatgg
      661 tgtctaaggg cgaagagctg atcaaggaaa atatgcgtat gaaggtggtc atggaaggtt
      721 cggtcaacgg ccaccaattc aaatgcacag gtgaaggaga aggcaatccg tacatgggaa
      781 ctcaaaccat gaggatcaaa gtcatcgagg gaggacccct gccatttgcc tttgacattc
      841 ttgccacgtc gttcatgtat ggcagccgta cttttatcaa gtacccgaaa ggcattcctg
      901 atttctttaa acagtccttt cctgagggtt ttacttggga aagagttacg agatacgaag
      961 atggtggagt cgtcaccgtc atgcaggaca ccagccttga ggatggctgt ctcgtttacc
     1021 acgtccaagt cagaggggta aactttccct ccaatggtcc cgtgatgcag aagaagacca
     1081 agggttggga gcctaataca gagatgatgt atccagcaga tggtggtctg aggggataca
     1141 ctcatatggc actgaaagtt gatggtggtg gccatctgtc ttgctctttc gtaacaactt
     1201 acaggtcaaa aaagaccgtc gggaacatca agatgcccgg tatccatgcc gttgatcacc
     1261 gcctggaaag gttagaggaa agtgacaatg aaatgttcgt agtacaacgc gaacacgcag
     1321 ttgccaagtt cgccgggctt ggtggtggga tggacgagct gtacaaggga agcggagcta
     1381 ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga cctatgaccg
     1441 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     1501 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     1561 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     1621 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     1681 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     1741 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     1801 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     1861 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacat
     1921 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     1981 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgacaacaga
     2041 gacctagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2101 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     2161 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     2221 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     2281 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     2341 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     2401 gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat
     2461 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     2521 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     2581 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     2641 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     2701 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     2761 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     2821 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     2881 tggagccggt gagcgtgggt atcgcggtat cattgcagca ctggggccag atggtaagcc
     2941 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     3001 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     3061 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     3121 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccact
//
