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

LOCUS       pGL3-NMB-Nicking/PegRNA-PGK-puroR-mRFP1               5674 bp DNA     circular SYN 07-NOV-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5674)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 21, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..5674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..55
                     /label=OL2640
     primer_bind     complement(21..44)
                     /label=OL459
     primer_bind     24..40
                     /label=OL1149
     primer_bind     36..60
                     /label=OL535
     misc_feature    41..115
                     /label=hCtRNA
     primer_bind     56..79
                     /label=OL464
     primer_bind     complement(96..135)
                     /label=OL541
     primer_bind     complement(96..116)
                     /label=OL543
     primer_bind     complement(98..116)
                     /label=OL465
     primer_bind     complement(101..115)
                     /label=OL2646
     primer_bind     complement(101..115)
                     /label=OL2648
     primer_bind     116..159
                     /label=OL542
     misc_feature    116..135
                     /label=NMB-nicking
     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..164
                     /label=OL2724
     primer_bind     136..151
                     /label=OL2647
     primer_bind     complement(194..230)
                     /label=OL463
     misc_feature    212..286
                     /label=hCtRNA
     primer_bind     212..231
                     /label=OL535
     primer_bind     212..226
                     /label=OL2640
     primer_bind     227..250
                     /label=OL464
     primer_bind     complement(267..306)
                     /label=OL543
     primer_bind     complement(267..287)
                     /label=OL541
     primer_bind     complement(269..287)
                     /label=OL465
     primer_bind     complement(272..292)
                     /label=OL2648
     primer_bind     complement(272..286)
                     /label=OL2646
     primer_bind     287..316
                     /label=OL544
     misc_feature    287..306
                     /label=NMB-Spacer
     primer_bind     306..335
                     /label=OL2724
     primer_bind     306..322
                     /label=OL2647
     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=OL542
     primer_bind     complement(365..385)
                     /label=OL463
     misc_feature    383..396
                     /label=RTT
     primer_bind     complement(387..416)
                     /label=OL545
     misc_feature    397..407
                     /label=PBS
     primer_bind     complement(399..425)
                     /label=OL1402
     primer_bind     complement(407..431)
                     /label=OL2650
     primer_bind     complement(407..431)
                     /label=OL2725
     primer_bind     complement(414..430)
                     /label=OL1148
     primer_bind     complement(414..430)
                     /label=OL2854
     misc_feature    451..568
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        617..1127
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1046..1064
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1149..1745
                     /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(1149..1168)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1645..1665
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1716..1770
                     /label=OL901
     primer_bind     complement(1731..1753)
                     /label=OL384
     primer_bind     1738..1769
                     /label=OL379
     CDS             1746..1802
                     /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"
     primer_bind     1746..1802
                     /label=OL866
     primer_bind     complement(1746..1802)
                     /label=OL867
     primer_bind     1783..1826
                     /label=OL902
     primer_bind     complement(1783..1810)
                     /label=OL966
     RBS             1785..1792
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1803..2480
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(2458..2505)
                     /label=OL903
     primer_bind     2479..2497
                     /label=OL965
     polyA_signal    2627..2748
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2638..2657)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2692..2711
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2996..3013)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3167..3755)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3247..3266)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3926..4786)
                     /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     4549..4568
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4787..4891)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4918..5373
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5005..5024)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5215..5236
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5504..5552
                     /note="synthetic polyadenylation signal"
     misc_feature    5566..5657
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5606..5625
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 agtgaacgga tctcgacggt atcgatcacg agactagcct agagggggta tagctcagtg
       61 gtagagcatt tgactgcaga tcaagaggtc cccggttcaa atccgggtgc ccccttcctt
      121 tccaggtcac ttcatgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
      181 tcaacttgaa aaagtggcac cgagtcggtg cagagggggt atagctcagt ggtagagcat
      241 ttgactgcag atcaagaggt ccccggttca aatccgggtg cccccttggt ccctcaggga
      301 ggtgtggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      361 aaaagtggca ccgagtcggt gcggacagct acccacacct ccctgagttt ttttctcgac
      421 ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
      481 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
      541 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact
      601 ttggccgcgg ctcgaggggg ttggggttgc gccttttcca aggcagccct gggtttgcgc
      661 agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggactc
      721 gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc
      781 ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt
      841 gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc
      901 agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag
      961 ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag
     1021 tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt
     1081 cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg
     1141 agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     1201 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     1261 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     1321 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     1381 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     1441 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     1501 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     1561 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     1621 ccttcctgga aacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     1681 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     1741 gtgccgctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac
     1801 ctatggcgag tagcgaagac gttatcaaag agttcatgcg tttcaaagtt cgtatggaag
     1861 gttccgttaa cggtcacgag ttcgaaatcg aaggtgaagg tgaaggtcgt ccgtacgaag
     1921 gtacccagac cgctaaactg aaagttacca aaggtggtcc gctgccgttc gcttgggaca
     1981 tcctgtcccc gcagttccag tacggttcca aagcttacgt taaacacccg gctgacatcc
     2041 cggactacct gaaactgtcc ttcccggaag gtttcaaatg ggaacgtgtt atgaacttcg
     2101 aagacggtgg tgttgttacc gttacccagg actcctccct gcaagacggt gagttcatct
     2161 acaaagttaa actgcgtggt accaacttcc cgtccgacgg tccggttatg cagaaaaaaa
     2221 ccatgggttg ggaagcttcc accgaacgta tgtacccgga agacggtgct ctgaaaggtg
     2281 aaatcaaaat gcgtctgaaa ctgaaagacg gtggtcacta cgacgctgaa gttaaaacca
     2341 cctacatggc taaaaaaccg gttcagctgc cgggtgctta caaaaccgac atcaaactgg
     2401 acatcacctc ccacaacgaa gactacacca tcgttgaaca gtacgaacgt gctgaaggtc
     2461 gtcactccac cggtgcttaa cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg
     2521 cacgacccca tgcatcggta cctttaagac caatgactta caaggcagct gtagatctta
     2581 gccactttct agagtcgggg cggccggccg cttcgagcag acatgataag atacattgat
     2641 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     2701 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     2761 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     2821 aacctctaca aatgtggtaa aatcgataag gatccgtcga ccgatgccct tgagagcctt
     2881 caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac
     2941 tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc
     3001 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3061 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3121 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3181 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3241 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3301 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3361 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3421 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3481 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3541 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3601 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3661 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3721 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3781 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3841 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3901 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3961 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4021 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgggac ccacgctcac
     4081 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4141 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4201 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4261 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4321 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4381 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4441 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4501 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4561 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4621 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4681 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4741 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4801 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4861 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     4921 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4981 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     5041 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     5101 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     5161 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     5221 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     5281 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     5341 cgaattttaa caaaatatta acgcttacaa tttgccattc gccattcagg ctgcgcaact
     5401 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagcccaag ctaccatgat
     5461 aagtaagtaa tattaaggta cgggaggtac ttggagcggc cgcaataaaa tatctttatt
     5521 ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat acgctctcca
     5581 tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg caagtgcagg
     5641 tgccagaaca tttctctatc gataggtacc gatt
//