LOCUS       pKG2387_pPV2-mRu        3176 bp    DNA     circular SYN 07-SEP-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 3176)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported May 7, 2025 from SnapGene 8.0.3
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3176
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     rep_origin      38..626
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     527..546
                     /label=oSEQ067 pBR322-ori_fwd
     primer_bind     627..650
                     /label=pPV2-puro_fwd
     primer_bind     627..644
                     /label=oKL179
     primer_bind     629..665
                     /label=oKL260
     primer_bind     complement(629..646)
                     /label=pPV1_bb_rev_v2
     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"
     primer_bind     657..674
                     /label=oCO03 mRuby2_fwd
     primer_bind     657..674
                     /label=oKL266
     primer_bind     complement(683..704)
                     /label=oEP076
     misc_feature    703..706
                     /label=splice site 1
     primer_bind     705..724
                     /label=oEP073
     primer_bind     727..746
                     /label=oSEQ062 mRuby2_fwd
     primer_bind     complement(727..746)
                     /label=oSEQ093 mRuby2_rev
     misc_feature    925..928
                     /label=splice site 2
     primer_bind     complement(1145..1164)
                     /label=oCO04 mRuby2_rev
     misc_feature    1203..1206
                     /label=splice site 3
     primer_bind     complement(1259..1278)
                     /label=oSEQ061 mRuby2_rev
     primer_bind     1259..1276
                     /label=oKL117
     primer_bind     1293..1312
                     /label=OSEQ091 mRuby2_fwd_SEQ
     primer_bind     complement(1347..1388)
                     /label=oKL303
                     /note="rev for PCR192"
     primer_bind     1368..1390
                     /label=oKL166
     primer_bind     1368..1389
                     /label=oKL141
     primer_bind     1368..1388
                     /label=oKL183
     primer_bind     complement(1368..1387)
                     /label=oKL182
     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"
     primer_bind     1414..1453
                     /label=oKL250
     primer_bind     1414..1436
                     /label=oKL266
     primer_bind     complement(1416..1433)
                     /label=oKL202
     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     1434..1455
                     /label=pPV2-puro_fwd
     primer_bind     complement(1434..1453)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1434..1453
                     /label=oSEQ047 phage-dest-downtream-seq
     primer_bind     complement(1899..1926)
                     /label=puro1_rev
     primer_bind     1907..1934
                     /label=puro2_fwd
     primer_bind     1930..1950
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1999..2014
                     /label=OSEQ086 Puro_rev_SEQ
     primer_bind     complement(2013..2050)
                     /label=oKL300
                     /note="for PCR188 P2A-PuroR"
     primer_bind     complement(2013..2030)
                     /label=oKL066
     primer_bind     complement(2013..2030)
                     /label=oKL251
     primer_bind     complement(2016..2057)
                     /label=pPV2-puro_rev
     primer_bind     2038..2058
                     /label=pPV1_bb_fwd_v2
     primer_bind     2038..2058
                     /label=pPV-bb-fwd-v2
     primer_bind     2038..2052
                     /label=pPV1_bb_fwd
     primer_bind     complement(2039..2058)
                     /label=oKL247
     promoter        2078..2182
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2115..2136)
                     /label=oKL157
     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     2183..2207
                     /label=oSEQ083 Amp-fwd
     primer_bind     complement(2401..2420)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(2401..2420)
                     /label=oSEQ066 Amp_rev
     primer_bind     complement(2597..2614)
                     /label=oKL048
     primer_bind     2597..2614
                     /label=oKL191
     primer_bind     complement(2604..2621)
                     /label=oKL070
     primer_bind     complement(2644..2663)
                     /label=oEP074
     primer_bind     2644..2663
                     /label=oEP075
     primer_bind     2881..2919
                     /label=Amp2-ori_fwd
     primer_bind     complement(2883..2900)
                     /label=AmpR1_rev
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
//
