LOCUS       sequence-479901-        6383 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Express SmBiT-PPP1R15A 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 6383)
  AUTHORS   Promega
  TITLE     Promega Plasmid Collection
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6383)
  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            "Unpublished"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: SmBiT-PPP1R15A Fusion Vector
FEATURES             Location/Qualifiers
     source          1..6383
                     /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    3446..3567
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3457..3476)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3682..3715)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3725..3772
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3804..4598
                     /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(3858..3877)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4468..4487
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4756..5344)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4836..4855)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5470..5518
                     /note="synthetic polyadenylation signal"
     CDS             complement(5542..5940)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      5937..5946
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5971..6328)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5985..6120
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6137..6328
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6285..6305
                     /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 cgctcaccat ggtgaccggc
     1201 taccggctgt tcgaggagat tctcgggagc tccggtggtg gcgggagcgg aggtggaggc
     1261 tcgagcggtg cgatcgccat ggccccaggc caagcacccc atcaggctac cccgtggagg
     1321 gatgcccacc ctttcttcct cctgtcccca gtgatgggcc tcctcagccg cgcctggagc
     1381 cgcctgaggg gcctgggacc tctagagccc tggctggtgg aagcagtaaa aggagcagct
     1441 ctggtagaag ctggcctgga gggagaagct aggactcctc tggcaatccc ccatacccct
     1501 tggggcagac gccctgaaga ggaggctgaa gacagtggag gccctggaga ggacagagaa
     1561 acactggggc tgaaaaccag cagttccctt cctgaagcct ggggactttt ggatgatgat
     1621 gatggcatgt atggtgagcg agaggcaacc agtgtcccta gagggcaggg aagtcaattt
     1681 gcagatggcc agcgtgctcc cctgtctccc agccttctga taaggacact gcaaggttct
     1741 gataagaacc caggggagga gaaagccgag gaagagggag ttgctgaaga ggagggagtt
     1801 aacaagttct cttatccacc atcacaccgg gagtgttgtc cagccgtgga ggaggaggac
     1861 gatgaagaag ctgtaaagaa agaagctcac agaacctcta cttctgcctt gtctccagga
     1921 tccaagccca gcacttgggt gtcttgccca ggggaggaag agaatcaagc cacggaggat
     1981 aaaagaacag aaagaagtaa aggagccagg aagacctccg tgtccccccg atcttcaggc
     2041 tccgacccca ggtcctggga gtatcgttca ggagaggcgt ccgaggagaa ggaggaaaag
     2101 gcacacaaag aaactgggaa aggagaagct gccccagggc cgcaatcctc agccccagcc
     2161 cagaggcccc agctcaagtc ctggtggtgc caacccagtg atgaagagga gggtgaggtc
     2221 aaggctttgg gggcagctga gaaggatgga gaagctgagt gtcctccctg catcccccca
     2281 ccaagtgcct tcctgaaggc ctgggtgtat tggccaggag aggacacaga ggaagaggaa
     2341 gatgaggaag aagatgagga cagtgactct ggatcagatg aggaagaggg agaagctgag
     2401 gcttcctctt ccactcctgc tacaggtgtc ttcttgaagt cctgggtcta tcagccagga
     2461 gaggacacag aggaggagga agatgaggac agtgatacag gatcagccga ggatgaaaga
     2521 gaagctgaga cttctgcttc cacaccccct gcaagtgctt tcttgaaggc ctgggtgtat
     2581 cggccaggag aggacacgga ggaggaggaa gatgaggatg tggatagtga ggataaggaa
     2641 gatgattcag aagcagcctt gggagaagct gagtcagacc cacatccctc ccacccggac
     2701 cagagggccc acttcagggg ctggggatat cgacctggaa aagagacaga ggaagaggaa
     2761 gctgctgagg actggggaga agctgagccc tgccccttcc gagtggccat ctatgtacct
     2821 ggagagaagc caccgcctcc ctgggctcct cctaggctgc ccctccgact gcaaaggcgg
     2881 ctcaagcgcc cagaaacccc tactcatgat ccggaccctg agactcccct aaaggccaga
     2941 aaggtgcgct tctccgagaa ggtcactgtc catttcctgg ctgtctgggc agggccggcc
     3001 caggccgccc gccagggccc ctgggagcag cttgctcggg atcgcagccg cttcgcacgc
     3061 cgcatcaccc aggcccagga ggagctgagc ccctgcctca cccctgctgc ccgggccaga
     3121 gcctgggcac gcctcaggaa cccaccttta gcccccatcc ctgccctcac ccagaccttg
     3181 ccttcctcct ctgtcccttc gtccccagtc cagaccacgc ccttgagcca agctgtggcc
     3241 acaccttccc gctcgtctgc tgctgcagcg gctgccctgg acctcagtgg gaggcgtggc
     3301 gtttaaacga attcgggctc ggtacccggg gatcctctag agtcgacctg caggcatgca
     3361 agctgatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     3421 gcaaggccgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact
     3481 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     3541 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     3601 gttcaggggg agatgtggga ggttttttta agcaagtaaa acctctacaa atgtggtaaa
     3661 atcttaatta agaattctaa tataacttcg tatagcatac attatacgaa gttatggatc
     3721 cactgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
     3781 gaactaaacc caggaggcag atcatgattg aacaagatgg attgcacgca ggttctccgg
     3841 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3901 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3961 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     4021 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     4081 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     4141 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     4201 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     4261 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     4321 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     4381 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     4441 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     4501 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     4561 gcatcgcctt ctatcgcctt cttgacgagt tcttctgatt cgaaatgacc gaccaagcga
     4621 cgcccaaccg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4681 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4741 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4801 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4861 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4921 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4981 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5041 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5101 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5161 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     5221 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5281 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatt
     5341 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5401 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttatagtc
     5461 cggaatcgtc acacaaaaaa ccaacacaca gatgtaatga aaataaagat attttattgc
     5521 ggccatcgtg atggctagag tttagccctc ccacacataa ccagagggca gcaattcacg
     5581 aatcccaact gccgtcggct gtccatcact gtccttcact atggctttga tcccaggatg
     5641 cagatcgaga agcacctgtc ggcaccgtcc gcaggggctc aagatgcccc tgttctcatt
     5701 tccgatcgcg acgatacaag tcaggttgcc agctgccgca gcagcagcag tgcccagcac
     5761 cacgagttct gcacaaggtc ccccagtaaa atgatataca ttgacaccag tgaagatgcg
     5821 gccgtcgcta gagagagctg cgctggcgac gctgtagtct tcagagatgg ggatgctgtt
     5881 gattgtagcc gttgctcttt caatgagggt ggattcttct tgagacaaag gcttggccat
     5941 ggtggcgcta gtgtcagaag aatcgagctt tttgcaaaag cctaggcctc caaaaaagcc
     6001 tcctcactac ttctggaata gctcagaggc cgaggcggcc tcggcctctg cataaataaa
     6061 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat
     6121 gggcggagtt aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact
     6181 tctgcctgct ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat
     6241 gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac
     6301 attccacagc tggttctttc cgcctcagaa ggtacctaac caagttcctc tttcagaggt
     6361 tatttcaggc catggtgctg cgc
//
