pGL3-U6-sgRNA-PGK-puroR-mRFP1

LOCUS       pGL3-U6-sgRNA-PGK-puroR-mRFP1              5683 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 5683)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 20, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..5683
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     72..92
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     242..265
                     /label=OL109
     primer_bind     243..262
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     295..318
                     /label=OL963
     misc_RNA        343..418
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(401..418)
                     /label=OL769
     primer_bind     426..448
                     /label=OL1049
     misc_feature    470..587
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        636..1146
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(730..749)
                     /label=OL1068
     primer_bind     920..936
                     /label=OL446
     primer_bind     1065..1083
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1168..1764
                     /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(1168..1187)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1664..1684
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1735..1789
                     /label=OL901
     primer_bind     complement(1741..1764)
                     /label=OL306
     primer_bind     complement(1750..1772)
                     /label=OL384
     primer_bind     1757..1788
                     /label=OL379
     CDS             1765..1821
                     /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     1765..1821
                     /label=OL866
     primer_bind     complement(1765..1821)
                     /label=OL867
     primer_bind     1802..1845
                     /label=OL902
     primer_bind     complement(1802..1829)
                     /label=OL966
     RBS             1804..1811
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1822..2499
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(2477..2524)
                     /label=OL903
     primer_bind     2498..2516
                     /label=OL965
     primer_bind     2500..2522
                     /label=OL305
     polyA_signal    2646..2767
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2657..2676)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2711..2730
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2956..2979)
                     /label=OL1043
     primer_bind     2957..2979
                     /label=OL1044
     primer_bind     complement(3015..3032)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3186..3774)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3266..3285)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3368..3387)
                     /label=OL1041
     primer_bind     complement(3501..3520)
                     /label=OL631
     primer_bind     3557..3576
                     /label=OL798
     primer_bind     complement(3798..3837)
                     /label=OL660
     primer_bind     3818..3837
                     /label=OL659
     CDS             complement(3945..4805)
                     /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     4568..4587
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     4721..4740
                     /label=OL1033
     primer_bind     complement(4721..4740)
                     /label=OL1034
     promoter        complement(4806..4910)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4937..5392
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5024..5043)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5234..5255
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5523..5571
                     /note="synthetic polyadenylation signal"
     misc_feature    5585..5676
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5625..5644
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
       61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacacc gtgagaccga gagagggtct cagttttaga gctagaaata
      361 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      421 tttttaaaga attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa
      481 aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca
      541 tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca
      601 gggacagcag agatccactt tggccgcggc tcgagggggt tggggttgcg ccttttccaa
      661 ggcagccctg ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc
      721 ggcgccgacc ctgggactcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc
      781 gccgctaccc ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt
      841 tccttgcggt tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc
      901 ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc
      961 aatagcggct gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag
     1021 gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc
     1081 aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct
     1141 ccccaggggg atccaccgga gcttaccatg accgagtaca agcccacggt gcgcctcgcc
     1201 acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc
     1261 gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa
     1321 ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc
     1381 gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc
     1441 ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc
     1501 ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg
     1561 cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc
     1621 gagcgcgccg gggtgcccgc cttcctggaa acctccgcgc cccgcaacct ccccttctac
     1681 gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg
     1741 tgcatgaccc gcaagcccgg tgccgctact aacttcagcc tgctgaagca ggctggagac
     1801 gtggaggaga accctggacc tatggcgagt agcgaagacg ttatcaaaga gttcatgcgt
     1861 ttcaaagttc gtatggaagg ttccgttaac ggtcacgagt tcgaaatcga aggtgaaggt
     1921 gaaggtcgtc cgtacgaagg tacccagacc gctaaactga aagttaccaa aggtggtccg
     1981 ctgccgttcg cttgggacat cctgtccccg cagttccagt acggttccaa agcttacgtt
     2041 aaacacccgg ctgacatccc ggactacctg aaactgtcct tcccggaagg tttcaaatgg
     2101 gaacgtgtta tgaacttcga agacggtggt gttgttaccg ttacccagga ctcctccctg
     2161 caagacggtg agttcatcta caaagttaaa ctgcgtggta ccaacttccc gtccgacggt
     2221 ccggttatgc agaaaaaaac catgggttgg gaagcttcca ccgaacgtat gtacccggaa
     2281 gacggtgctc tgaaaggtga aatcaaaatg cgtctgaaac tgaaagacgg tggtcactac
     2341 gacgctgaag ttaaaaccac ctacatggct aaaaaaccgg ttcagctgcc gggtgcttac
     2401 aaaaccgaca tcaaactgga catcacctcc cacaacgaag actacaccat cgttgaacag
     2461 tacgaacgtg ctgaaggtcg tcactccacc ggtgcttaac gcccgcccca cgacccgcag
     2521 cgcccgaccg aaaggagcgc acgaccccat gcatcggtac ctttaagacc aatgacttac
     2581 aaggcagctg tagatcttag ccactttcta gagtcggggc ggccggccgc ttcgagcaga
     2641 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     2701 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     2761 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     2821 ggttttttaa agcaagtaaa acctctacaa atgtggtaaa atcgataagg atccgtcgac
     2881 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
     2941 tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag
     3001 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3061 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3121 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3181 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3241 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3301 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3361 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3421 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3481 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3541 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3601 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3661 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3721 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3781 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3841 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3901 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3961 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4021 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4081 ccgcgggacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4141 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4201 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4261 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4321 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4381 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4441 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4501 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4561 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4621 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4681 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4741 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4801 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4861 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4921 cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4981 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     5041 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5101 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     5161 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5221 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     5281 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5341 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttgccattcg
     5401 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     5461 cagcccaagc taccatgata agtaagtaat attaaggtac gggaggtact tggagcggcc
     5521 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag
     5581 tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca aaataggctg
     5641 tccccagtgc aagtgcaggt gccagaacat ttctctatcg ata
//