pGL3-KRAS-Nicking/PegRNA-PGK-puroR-mRFP1

LOCUS       pGL3-KRAS-Nicking/PegRNA-PGK-puroR-mRFP1               5670 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 5670)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 22, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..5670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..61
                     /label=OL2745
     misc_feature    41..115
                     /label=hCtRNA
     primer_bind     56..79
                     /label=OL464
     primer_bind     complement(96..135)
                     /label=OL3436
     primer_bind     complement(96..116)
                     /label=OL3438
     primer_bind     complement(98..115)
                     /label=OL465
     primer_bind     116..159
                     /label=OL3437
     misc_feature    116..135
                     /label=KRAS-nickingRNA
     primer_bind     135..176
                     /label=OL462
     misc_RNA        136..211
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     136..159
                     /label=OL3439
     primer_bind     complement(192..212)
                     /label=OL3440
     primer_bind     complement(194..230)
                     /label=OL463
     misc_feature    212..286
                     /label=hCtRNA
     primer_bind     212..232
                     /label=OL2745
     primer_bind     227..250
                     /label=OL464
     primer_bind     complement(267..306)
                     /label=OL3438
     primer_bind     complement(267..287)
                     /label=OL3436
     primer_bind     complement(269..286)
                     /label=OL465
     primer_bind     287..330
                     /label=OL3439
     misc_feature    287..306
                     /label=KRAS-Spacer
     misc_RNA        307..382
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     307..347
                     /label=OL462
     primer_bind     307..330
                     /label=OL3437
     primer_bind     complement(363..428)
                     /label=OL3440
     primer_bind     complement(365..383)
                     /label=OL463
     misc_feature    383..394
                     /label=RTT
     misc_feature    395..403
                     /label=PBS
     misc_feature    447..564
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        613..1123
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1042..1060
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1145..1741
                     /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(1145..1164)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1641..1661
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1742..1798
                     /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             1781..1788
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1799..2476
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(2454..2501)
                     /label=OL903
     primer_bind     2475..2493
                     /label=OL965
     polyA_signal    2623..2744
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2634..2653)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2688..2707
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2992..3009)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3163..3751)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3243..3262)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3922..4782)
                     /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     4545..4564
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4783..4887)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4914..5369
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5001..5020)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5211..5232
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5500..5548
                     /note="synthetic polyadenylation signal"
     misc_feature    5562..5653
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5602..5621
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 agtgaacgga tctcgacggt atcgatcacg agactagcct agagggggta tagctcagtg
       61 gtagagcatt tgactgcaga tcaagaggtc cccggttcaa atccgggtgc cccctacaag
      121 atttacctct attgtgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
      181 tcaacttgaa aaagtggcac cgagtcggtg cagagggggt atagctcagt ggtagagcat
      241 ttgactgcag atcaagaggt ccccggttca aatccgggtg ccccctaaac ttgtggtagt
      301 tggagcgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      361 aaaagtggca ccgagtcggt gcacgccatc agctccaact accttttttt ctcgacctcg
      421 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
      481 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
      541 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccactttgg
      601 ccgcggctcg agggggttgg ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg
      661 acgcggctgc tctgggcgtg gttccgggaa acgcagcggc gccgaccctg ggactcgcac
      721 attcttcacg tccgttcgca gcgtcacccg gatcttcgcc gctacccttg tgggcccccc
      781 ggcgacgctt cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg
      841 gacgtgacaa acggaagccg cacgtctcac tagtaccctc gcagacggac agcgccaggg
      901 agcaatggca gcgcgccgac cgcgatgggc tgtggccaat agcggctgct cagcagggcg
      961 cgccgagagc agcggccggg aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg
     1021 ggccctgttc ctgcccgcgc ggtgttccgc attctgcaag cctccggagc gcacgtcggc
     1081 agtcggctcc ctcgttgacc gaatcaccga cctctctccc cagggggatc caccggagct
     1141 taccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     1201 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     1261 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     1321 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     1381 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     1441 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     1501 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct
     1561 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     1621 cctggaaacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     1681 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     1741 cgctactaac ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc ctggacctat
     1801 ggcgagtagc gaagacgtta tcaaagagtt catgcgtttc aaagttcgta tggaaggttc
     1861 cgttaacggt cacgagttcg aaatcgaagg tgaaggtgaa ggtcgtccgt acgaaggtac
     1921 ccagaccgct aaactgaaag ttaccaaagg tggtccgctg ccgttcgctt gggacatcct
     1981 gtccccgcag ttccagtacg gttccaaagc ttacgttaaa cacccggctg acatcccgga
     2041 ctacctgaaa ctgtccttcc cggaaggttt caaatgggaa cgtgttatga acttcgaaga
     2101 cggtggtgtt gttaccgtta cccaggactc ctccctgcaa gacggtgagt tcatctacaa
     2161 agttaaactg cgtggtacca acttcccgtc cgacggtccg gttatgcaga aaaaaaccat
     2221 gggttgggaa gcttccaccg aacgtatgta cccggaagac ggtgctctga aaggtgaaat
     2281 caaaatgcgt ctgaaactga aagacggtgg tcactacgac gctgaagtta aaaccaccta
     2341 catggctaaa aaaccggttc agctgccggg tgcttacaaa accgacatca aactggacat
     2401 cacctcccac aacgaagact acaccatcgt tgaacagtac gaacgtgctg aaggtcgtca
     2461 ctccaccggt gcttaacgcc cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg
     2521 accccatgca tcggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
     2581 ctttctagag tcggggcggc cggccgcttc gagcagacat gataagatac attgatgagt
     2641 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     2701 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     2761 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     2821 tctacaaatg tggtaaaatc gataaggatc cgtcgaccga tgcccttgag agccttcaac
     2881 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc
     2941 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt cctcgctcac
     3001 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3061 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3121 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3181 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3241 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3301 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3361 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3421 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3481 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3541 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3601 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3661 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3721 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3781 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3841 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3901 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3961 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4021 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgggacccac gctcaccggc
     4081 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4141 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4201 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     4261 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4321 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4381 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4441 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4501 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4561 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4621 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4681 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     4741 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     4801 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4861 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc
     4921 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4981 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     5041 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     5101 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     5161 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     5221 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     5281 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5341 ttttaacaaa atattaacgc ttacaatttg ccattcgcca ttcaggctgc gcaactgttg
     5401 ggaagggcga tcggtgcggg cctcttcgct attacgccag cccaagctac catgataagt
     5461 aagtaatatt aaggtacggg aggtacttgg agcggccgca ataaaatatc tttattttca
     5521 ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac taacatacgc tctccatcaa
     5581 aacaaaacga aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag tgcaggtgcc
     5641 agaacatttc tctatcgata ggtaccgatt
//