LOCUS       sequence-393400-        5684 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mRFP and T2A linker are inserted in between the hPGK promoter and 
            the puromycin resistance gene (PuroR) on pGL3-U6-sgRNA-PGK-puromycin
            to allow simultaneous monitoring and enrichment of transfected ho.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5684)
  AUTHORS   Ayo TE, Xu H
  TITLE     Generating a New sgRNA Vector, pGL3-U6-sgRNA-PGK-mRFP-T2A-PuroR, to 
            Improve Base Editing.
  JOURNAL   J Genome Ed Regul. 2022;2:246146. doi: 10.32371/jger/246146. Epub 
            2022 Dec 17.
  PUBMED    38076397
REFERENCE   2  (bases 1 to 5684)
  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: "J Genome Ed
            Regul."; date: "2022"; volume: "2:246146. doi"; pages: " 
            10.32371/jger/246146. Epub 2022 Dec 17"
COMMENT     Sequence Label: pGL3-U6-sgRNA-PGK- mRFP-T2A-PuroR
FEATURES             Location/Qualifiers
     source          1..5684
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    103..224
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(114..133)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     168..187
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(472..489)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(643..1231)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(723..742)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1402..2193,2194..2262))
                     /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     2025..2044
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2263..2367)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2394..2849
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2481..2500)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2691..2712
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    2980..3028
                     /note="synthetic polyadenylation signal"
     misc_feature    3042..3133
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     3082..3101
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        3212..3460
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3212..3232
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3383..3402
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3484..3559
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    3611..3728
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3777..4287
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4206..4224
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4987..5040
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5041..5640
                     /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(5041..5060)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5537..5557
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 accccatgca tcggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
       61 ctttctagag tcggggcggc cggccgcttc gagcagacat gataagatac attgatgagt
      121 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
      181 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
      241 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
      301 tctacaaatg tggtaaaatc gataaggatc cgtcgaccga tgcccttgag agccttcaac
      361 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc
      421 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt cctcgctcac
      481 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
      541 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
      601 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
      661 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
      721 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
      781 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
      841 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
      901 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
      961 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1021 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     1081 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     1141 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     1201 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     1261 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     1321 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     1381 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     1441 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     1501 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgggacccac gctcaccggc
     1561 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     1621 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     1681 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     1741 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     1801 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     1861 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     1921 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     1981 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     2041 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     2101 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     2161 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     2221 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     2281 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     2341 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc
     2401 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2461 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2521 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2581 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2641 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2701 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2761 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2821 ttttaacaaa atattaacgc ttacaatttg ccattcgcca ttcaggctgc gcaactgttg
     2881 ggaagggcga tcggtgcggg cctcttcgct attacgccag cccaagctac catgataagt
     2941 aagtaatatt aaggtacggg aggtacttgg agcggccgca ataaaatatc tttattttca
     3001 ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac taacatacgc tctccatcaa
     3061 aacaaaacga aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag tgcaggtgcc
     3121 agaacatttc tctatcgata ggtaccgatt agtgaacgga tctcgacggt atcgatcacg
     3181 agactagcct cgagcggccg cccccttcac cgagggccta tttcccatga ttccttcata
     3241 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     3301 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     3361 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     3421 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggtgagaccg agagagggtc
     3481 tcagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa
     3541 agtggcaccg agtcggtgct ttttttaaag aattctcgac ctcgagacaa atggcagtat
     3601 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag
     3661 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc
     3721 aaaattttcg ggtttattac agggacagca gagatccact ttggccgcgg ctcgaggggg
     3781 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
     3841 cgtggttccg ggaaacgcag cggcgccgac cctgggactc gcacattctt cacgtccgtt
     3901 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
     3961 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
     4021 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
     4081 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
     4141 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
     4201 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
     4261 gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat ggccagctcc
     4321 gaggatgtca tcaaagagtt tatgagattt aaggtcaaga tggagggaag cgtcaacgga
     4381 cacgagttcg agattgaggg agaaggagaa ggccggcctt acgagggcac acaaaccgct
     4441 aagctcaagg tcacaaaagg aggacccctc cccttctcct gggatattct gagccctcag
     4501 ttccagtacg gaagcaaagc ctatgttaaa caccctgccg acatccctga ctatctgaag
     4561 ctctccttcc ctgaaggctt caagtgggag agattcatga acttcgagga cggaggcgtg
     4621 gtgacagtca cacaagatag caccctccag gacggagagt ttatttataa ggtgaaactc
     4681 agaggaacca acttcccctc cgatggccct gtcatgcaaa aaaaaacaat gggatgggaa
     4741 gcctccaccg agagaatgta tcctgaggat ggcgctctga aaggcgaaat taaaatgaga
     4801 ctgaaactca aagacggagg acactacgat gccgaggtca aaacaaccta caaggccaag
     4861 aaacaagtgc agctgcctgg cgcctacatg actgatatta aactcgacat tatcagccat
     4921 aatggggact acaccatcgt ggaacaatat gagagagctg agggcagaca tagcacaggc
     4981 gctggagagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct
     5041 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc cagggccgta
     5101 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgatccggac
     5161 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
     5221 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
     5281 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
     5341 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
     5401 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
     5461 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
     5521 gaaacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
     5581 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga
     5641 cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacg
//
