pGL3-RNF2-24ins-Nicking/PegRNA-PGK-puroR-mRFP1

LOCUS       pGL3-RNF2-24ins-Nicking/PegRNA-PGK-puroR-mRFP1                5711 bp DNA     circular SYN 08-NOV-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5711)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 22, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..5711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     37..61
                     /label=OL1080
     misc_feature    41..115
                     /label=hCtRNA
     primer_bind     56..79
                     /label=OL464
     primer_bind     complement(98..115)
                     /label=OL465
     primer_bind     complement(101..125)
                     /label=OL4232
     primer_bind     complement(101..119)
                     /label=OL4234
     misc_feature    116..136
                     /label=RNF2-nickingRNA
     primer_bind     122..151
                     /label=OL4233
     primer_bind     136..177
                     /label=OL462
     misc_RNA        137..212
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     137..152
                     /label=OL4235
     primer_bind     complement(195..231)
                     /label=OL463
     primer_bind     complement(197..216)
                     /label=OL4236
     misc_feature    213..287
                     /label=hCtRNA
     primer_bind     213..233
                     /label=OL1080
     primer_bind     228..251
                     /label=OL464
     primer_bind     complement(270..287)
                     /label=OL465
     primer_bind     complement(273..298)
                     /label=OL4234
     primer_bind     complement(273..291)
                     /label=OL4232
     misc_feature    288..307
                     /label=RNF2-spacer
     primer_bind     295..323
                     /label=OL4235
     misc_RNA        308..383
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     308..348
                     /label=OL462
     primer_bind     complement(366..387)
                     /label=OL463
     primer_bind     complement(368..404)
                     /label=OL4236
     misc_feature    384..429
                     /label=RTT
     primer_bind     401..449
                     /label=OL4237
     primer_bind     complement(405..453)
                     /label=OL4238
     misc_feature    430..444
                     /label=PBS
     misc_feature    488..605
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        654..1164
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1083..1101
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1186..1782
                     /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(1186..1205)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1682..1702
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1783..1839
                     /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"
     RBS             1822..1829
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1840..2517
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(2495..2542)
                     /label=OL903
     primer_bind     2516..2534
                     /label=OL965
     polyA_signal    2664..2785
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2675..2694)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2729..2748
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3033..3050)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3204..3792)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3284..3303)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3963..4823)
                     /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     4586..4605
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4824..4928)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4955..5410
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5042..5061)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5252..5273
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5541..5589
                     /note="synthetic polyadenylation signal"
     misc_feature    5603..5694
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5643..5662
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 agtgaacgga tctcgacggt atcgatcacg agactagcct agagggggta tagctcagtg
       61 gtagagcatt tgactgcaga tcaagaggtc cccggttcaa atccgggtgc cccctgtcaa
      121 ccattaagca aaacatgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
      181 atcaacttga aaaagtggca ccgagtcggt gcagaggggg tatagctcag tggtagagca
      241 tttgactgca gatcaagagg tccccggttc aaatccgggt gccccctgtc atcttagtca
      301 ttacctggtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg
      361 aaaaagtggc accgagtcgg tgctgagtta caacgaacac ctcagcttat cgtcgtcatc
      421 cttgtaatcg taatgactaa gatgtttttt tctcgacctc gagacaaatg gcagtattca
      481 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
      541 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
      601 attttcgggt ttattacagg gacagcagag atccactttg gccgcggctc gagggggttg
      661 gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt
      721 ggttccggga aacgcagcgg cgccgaccct gggactcgca cattcttcac gtccgttcgc
      781 agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg
      841 cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc
      901 gcacgtctca ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga
      961 ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg
     1021 gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg
     1081 cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac
     1141 cgaatcaccg acctctctcc ccagggggat ccaccggagc ttaccatgac cgagtacaag
     1201 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     1261 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     1321 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     1381 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     1441 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     1501 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     1561 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     1621 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggaaac ctccgcgccc
     1681 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     1741 gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccgctactaa cttcagcctg
     1801 ctgaagcagg ctggagacgt ggaggagaac cctggaccta tggcgagtag cgaagacgtt
     1861 atcaaagagt tcatgcgttt caaagttcgt atggaaggtt ccgttaacgg tcacgagttc
     1921 gaaatcgaag gtgaaggtga aggtcgtccg tacgaaggta cccagaccgc taaactgaaa
     1981 gttaccaaag gtggtccgct gccgttcgct tgggacatcc tgtccccgca gttccagtac
     2041 ggttccaaag cttacgttaa acacccggct gacatcccgg actacctgaa actgtccttc
     2101 ccggaaggtt tcaaatggga acgtgttatg aacttcgaag acggtggtgt tgttaccgtt
     2161 acccaggact cctccctgca agacggtgag ttcatctaca aagttaaact gcgtggtacc
     2221 aacttcccgt ccgacggtcc ggttatgcag aaaaaaacca tgggttggga agcttccacc
     2281 gaacgtatgt acccggaaga cggtgctctg aaaggtgaaa tcaaaatgcg tctgaaactg
     2341 aaagacggtg gtcactacga cgctgaagtt aaaaccacct acatggctaa aaaaccggtt
     2401 cagctgccgg gtgcttacaa aaccgacatc aaactggaca tcacctccca caacgaagac
     2461 tacaccatcg ttgaacagta cgaacgtgct gaaggtcgtc actccaccgg tgcttaacgc
     2521 ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgc atcggtacct
     2581 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttctaga gtcggggcgg
     2641 ccggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     2701 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     2761 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     2821 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat
     2881 cgataaggat ccgtcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     2941 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     3001 taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     3061 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     3121 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3181 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3241 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3301 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3361 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3421 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3481 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3541 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3601 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3661 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3721 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3781 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3841 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     3901 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     3961 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4021 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4081 cccagtgctg caatgatacc gcgggaccca cgctcaccgg ctccagattt atcagcaata
     4141 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4201 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     4261 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     4321 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     4381 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     4441 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     4501 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     4561 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     4621 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     4681 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     4741 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     4801 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     4861 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     4921 gttccgcgca catttccccg aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc
     4981 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     5041 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5101 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5161 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5221 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5281 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5341 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5401 cttacaattt gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     5461 gcctcttcgc tattacgcca gcccaagcta ccatgataag taagtaatat taaggtacgg
     5521 gaggtacttg gagcggccgc aataaaatat ctttattttc attacatctg tgtgttggtt
     5581 ttttgtgtga atcgatagta ctaacatacg ctctccatca aaacaaaacg aaacaaaaca
     5641 aactagcaaa ataggctgtc cccagtgcaa gtgcaggtgc cagaacattt ctctatcgat
     5701 aggtaccgat t
//