LOCUS       sequence-392769-        5904 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A pEF-BOS-EX-derived expression vector that contains an SV40 
            promoter-driven puromycin resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5904)
  AUTHORS   Fujii T, Inoue N, Nobeyama T, Inoue J, Ogasawara S, Otani Y, Fujii 
            S, Ito C, Fukunaga R
  TITLE     A simple, rapid, and efficient method for generating 
            multigene-knockout culture cells by the CRISPR/Cas9 system.
  JOURNAL   Genes Cells. 2023 Mar 2. doi: 10.1111/gtc.13021.
  PUBMED    36861495
REFERENCE   2  (bases 1 to 5904)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Genes 
            Cells. 2023 Mar 2. doi: 10.1111/gtc.13021."
COMMENT     Sequence Label: pUREF-EX
FEATURES             Location/Qualifiers
     source          1..5904
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..28
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        96..200
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(201..269,270..1061)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(419..438)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1232..1820
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1721..1740
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1974..1991
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2108..2129
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2144..2149,2150..2167,2168..2174)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2182..2198
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2187..2209
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2206..2222
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2206..2222
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2254..2464
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        2276..2472
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2323..2458
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        2559..3737
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2790..3728
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3685..3705
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(4422..4439)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4422..4438)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4431..4453)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4570..4590)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4575..4904
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4755..4890
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4993..5592
                     /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(4993..5012)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5489..5509
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    join(5822..5904,1..52)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5859..5878)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tctcccgatc
       61 cgtcgacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
      121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
      181 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
      241 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
      301 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
      361 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
      421 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
      481 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
      541 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
      601 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
      661 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
      721 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
      781 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
      841 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
      901 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
      961 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     1021 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     1081 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     1141 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     1201 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     1261 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     1321 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     1381 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     1441 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     1501 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     1561 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1621 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1681 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1741 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1801 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1861 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     1921 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     1981 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     2041 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     2101 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     2161 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     2221 accatgatta cgccaagctt aattccctcc ccagcaggca gaagtatgca aagcatgcat
     2281 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     2341 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     2401 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     2461 ggcttttgca aaaagctttg caaagatgga taaagtttta aacagagagg aatctttgca
     2521 gctaatggac cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg
     2581 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
     2641 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
     2701 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
     2761 ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg
     2821 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg
     2881 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
     2941 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
     3001 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
     3061 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
     3121 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
     3181 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
     3241 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg
     3301 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
     3361 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
     3421 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
     3481 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
     3541 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     3601 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     3661 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     3721 catttcaggt gtcgtgagga attcggtacc gcggccgccc ggggatccgc tagcgtcgac
     3781 tctagagtga gggtccccac ctgggaccct tgagagtatc aggtctccca cgtgggagac
     3841 aagaaatccc tgtttaatat ttaaacagca gtgttcccca tctgggtcct tgcacccctc
     3901 actctggcct cagccgactg cacagcggcc cctgcatccc cttggctgtg aggcccctgg
     3961 acaagcagag gtggccagag ctgggaggca tggccctggg gtcccacgaa tttgctgggg
     4021 aatctcgttt ttcttcttaa gacttttggg acatggtttg actcccgaac atcaccgacg
     4081 tgtctcctgt ttttctgggt ggcctcggga cacctgccct gcccccacga gggtcaggac
     4141 tgtgactctt tttagggcca ggcaggtgcc tggacatttg ccttgctgga cggggactgg
     4201 ggatgtggga gggagcagac aggaggaatc atgtcaggcc tgtgtgtgaa aggaagctcc
     4261 actgtcaccc tccacctctt caccccccac tcaccagtgt cccctccact gtcacattgt
     4321 aactgaactt caggataata aagwrwwwrm mwmmaaaaaa aaaaaaaaaa aaaaaaaaaa
     4381 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa anccgaattt cactggccgt cgttttacaa
     4441 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     4501 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc ctgcaggcat atggatatca
     4561 gatccctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     4621 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     4681 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     4741 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     4801 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     4861 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctt gcatgccggt
     4921 cggccgccac gaccggtgcc gccaccatcc cctgacccac gcccctgacc cctcacaagg
     4981 agacgacctt ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc
     5041 ccccgggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc
     5101 gtcgacccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc
     5161 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg
     5221 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc
     5281 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac
     5341 cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc
     5401 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg
     5461 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc
     5521 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag
     5581 cccggtgcct gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc
     5641 catggctccg accgaagcca cccggggcgg ccccgccgac cccgcacccg cccccgaggc
     5701 ccaccgactc tagaggatca taatcagcca taccacattt gtagaggttt tacttgcttt
     5761 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     5821 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     5881 aaataaagca tttttttcac tgca
//
