LOCUS       sequence-479900-        6324 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express PPP1R15A -SmBiT Fusion Protein in Mammalian Cells under a 
            CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6324)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6324)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: PPP1R15A -SmBiT Fusion Vector
FEATURES             Location/Qualifiers
     source          1..6324
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    24..105
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        338..641
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        642..845
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     796..816
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          981..1113
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1096..1115
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1157..1176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1157..1175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3387..3508
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3398..3417)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3623..3656)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3666..3713
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3745..4539
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3799..3818)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4409..4428
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4697..5285)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4777..4796)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5411..5459
                     /note="synthetic polyadenylation signal"
     CDS             complement(5483..5881)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5878..5887
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5912..6269)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5926..6061
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6078..6269
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6226..6246
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 aagacgcgtt aattaagacg tcggcagtga aaaaaatgct ttatttgtga aatttgtgat
       61 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
      121 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
      181 ctctacaaat gtggtatggc tgattatgat ccgcggccgc aatacgtcga cgttatcagc
      241 tgacttcgta cgagagccta ggattatggc gcgccactag ttattaatag taatcaatta
      301 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      361 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      421 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      481 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca
      541 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta
      601 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      661 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      721 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata
      781 accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      841 gagctggttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt tatcacagtt
      901 aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc tgcagaagtt
      961 ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat
     1021 agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc
     1081 ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccagttc aattacagct
     1141 cttaaggcta gagtattaat acgactcact atagggctag cgatcgccat ggccccaggc
     1201 caagcacccc atcaggctac cccgtggagg gatgcccacc ctttcttcct cctgtcccca
     1261 gtgatgggcc tcctcagccg cgcctggagc cgcctgaggg gcctgggacc tctagagccc
     1321 tggctggtgg aagcagtaaa aggagcagct ctggtagaag ctggcctgga gggagaagct
     1381 aggactcctc tggcaatccc ccatacccct tggggcagac gccctgaaga ggaggctgaa
     1441 gacagtggag gccctggaga ggacagagaa acactggggc tgaaaaccag cagttccctt
     1501 cctgaagcct ggggactttt ggatgatgat gatggcatgt atggtgagcg agaggcaacc
     1561 agtgtcccta gagggcaggg aagtcaattt gcagatggcc agcgtgctcc cctgtctccc
     1621 agccttctga taaggacact gcaaggttct gataagaacc caggggagga gaaagccgag
     1681 gaagagggag ttgctgaaga ggagggagtt aacaagttct cttatccacc atcacaccgg
     1741 gagtgttgtc cagccgtgga ggaggaggac gatgaagaag ctgtaaagaa agaagctcac
     1801 agaacctcta cttctgcctt gtctccagga tccaagccca gcacttgggt gtcttgccca
     1861 ggggaggaag agaatcaagc cacggaggat aaaagaacag aaagaagtaa aggagccagg
     1921 aagacctccg tgtccccccg atcttcaggc tccgacccca ggtcctggga gtatcgttca
     1981 ggagaggcgt ccgaggagaa ggaggaaaag gcacacaaag aaactgggaa aggagaagct
     2041 gccccagggc cgcaatcctc agccccagcc cagaggcccc agctcaagtc ctggtggtgc
     2101 caacccagtg atgaagagga gggtgaggtc aaggctttgg gggcagctga gaaggatgga
     2161 gaagctgagt gtcctccctg catcccccca ccaagtgcct tcctgaaggc ctgggtgtat
     2221 tggccaggag aggacacaga ggaagaggaa gatgaggaag aagatgagga cagtgactct
     2281 ggatcagatg aggaagaggg agaagctgag gcttcctctt ccactcctgc tacaggtgtc
     2341 ttcttgaagt cctgggtcta tcagccagga gaggacacag aggaggagga agatgaggac
     2401 agtgatacag gatcagccga ggatgaaaga gaagctgaga cttctgcttc cacaccccct
     2461 gcaagtgctt tcttgaaggc ctgggtgtat cggccaggag aggacacgga ggaggaggaa
     2521 gatgaggatg tggatagtga ggataaggaa gatgattcag aagcagcctt gggagaagct
     2581 gagtcagacc cacatccctc ccacccggac cagagggccc acttcagggg ctggggatat
     2641 cgacctggaa aagagacaga ggaagaggaa gctgctgagg actggggaga agctgagccc
     2701 tgccccttcc gagtggccat ctatgtacct ggagagaagc caccgcctcc ctgggctcct
     2761 cctaggctgc ccctccgact gcaaaggcgg ctcaagcgcc cagaaacccc tactcatgat
     2821 ccggaccctg agactcccct aaaggccaga aaggtgcgct tctccgagaa ggtcactgtc
     2881 catttcctgg ctgtctgggc agggccggcc caggccgccc gccagggccc ctgggagcag
     2941 cttgctcggg atcgcagccg cttcgcacgc cgcatcaccc aggcccagga ggagctgagc
     3001 ccctgcctca cccctgctgc ccgggccaga gcctgggcac gcctcaggaa cccaccttta
     3061 gcccccatcc ctgccctcac ccagaccttg ccttcctcct ctgtcccttc gtccccagtc
     3121 cagaccacgc ccttgagcca agctgtggcc acaccttccc gctcgtctgc tgctgcagcg
     3181 gctgccctgg acctcagtgg gaggcgtggc gtttctcaag gcagttcagg tggtggcggg
     3241 agcggaggtg gaggctcgag cggtgtgacc ggctaccggc tgttcgagga gattctgtaa
     3301 ggccgcgact ctagagtcga cctgcaggca tgcaagctga tttggctgct gccaccgctg
     3361 agcaaggccg cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac
     3421 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     3481 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     3541 ggttcagggg gagatgtggg aggttttttt aagcaagtaa aacctctaca aatgtggtaa
     3601 aatcttaatt aagaattcta atataacttc gtatagcata cattatacga agttatggat
     3661 ccactgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga
     3721 ggaactaaac ccaggaggca gatcatgatt gaacaagatg gattgcacgc aggttctccg
     3781 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3841 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3901 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     3961 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4021 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4081 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4141 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4201 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4261 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4321 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4381 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4441 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4501 cgcatcgcct tctatcgcct tcttgacgag ttcttctgat tcgaaatgac cgaccaagcg
     4561 acgcccaacc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4621 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4681 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4741 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4801 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4861 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4921 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4981 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5041 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5101 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     5161 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5221 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5281 ttcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5341 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttatagt
     5401 ccggaatcgt cacacaaaaa accaacacac agatgtaatg aaaataaaga tattttattg
     5461 cggccatcgt gatggctaga gtttagccct cccacacata accagagggc agcaattcac
     5521 gaatcccaac tgccgtcggc tgtccatcac tgtccttcac tatggctttg atcccaggat
     5581 gcagatcgag aagcacctgt cggcaccgtc cgcaggggct caagatgccc ctgttctcat
     5641 ttccgatcgc gacgatacaa gtcaggttgc cagctgccgc agcagcagca gtgcccagca
     5701 ccacgagttc tgcacaaggt cccccagtaa aatgatatac attgacacca gtgaagatgc
     5761 ggccgtcgct agagagagct gcgctggcga cgctgtagtc ttcagagatg gggatgctgt
     5821 tgattgtagc cgttgctctt tcaatgaggg tggattcttc ttgagacaaa ggcttggcca
     5881 tggtggcgct agtgtcagaa gaatcgagct ttttgcaaaa gcctaggcct ccaaaaaagc
     5941 ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct gcataaataa
     6001 aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt aggggcggga
     6061 tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg ctttgcatac
     6121 ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa ttgagatgca
     6181 tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct aactgacaca
     6241 cattccacag ctggttcttt ccgcctcaga aggtacctaa ccaagttcct ctttcagagg
     6301 ttatttcagg ccatggtgct gcgc
//
