LOCUS       sequence-487504-        4068 bp    DNA     circular SYN 30-SEP-2025
DEFINITION  spCas9 gRNA is driven by human U6 promoter, with the gRNA cassette 
            between PuroR and pA, making it detectable in polyA-based RNA-seq..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4068)
  TITLE     pb-Puro-gRNA-pA plasmid - gRNA is readable in RNAseq
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4068)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 30, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: PB-Puro-U6-gRNA-pA plasmid
FEATURES             Location/Qualifiers
     source          1..4068
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    835..856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(871..876,877..894,895..901)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    909..925
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     914..936
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     933..949
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     933..949
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   984..1018
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    1032..1153
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1043..1062)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1097..1116
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_RNA        complement(1180..1255)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1274..1522)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1332..1351)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1502..1522)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(1533..2132)
                     /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(1616..1636)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2113..2132
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(2401..2418)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2401..2417)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2410..2432)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2626..2645)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2745..2767
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2805..2823)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2891..2995
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2996..3064,3065..3856)
                     /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     complement(3214..3233)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4027..4068,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaacag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      781 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      841 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      901 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      961 caagctcggc gcgccattgg gatccctaga aagatagtct gcgtaaaatt gacgcatggc
     1021 tcttctagac gtaagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     1081 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     1141 caataaacaa gttttaatta aaagctctaa aacaaaaaag caccgactcg gtgccacttt
     1201 ttcaagttga taacggacta gccttatttt aacttgctat ttctagctct aaaacaggtc
     1261 ttctcgaaga cccggtgttt cgtcctttcc acaagatata taaagccaag aaatcgaaat
     1321 actttcaagt tacggtaagc atatgatagt ccattttaaa acataatttt aaaactgcaa
     1381 actacccaag aaattattac tttctacgtc acgtattttg tactaatatc tttgtgttta
     1441 cagtcaaatt aattccaatt atctctctaa cagccttgta tcgtatatgc aaatatgaag
     1501 gaatcatggg aaataggccc tcgtcctcga gctcaggcac cgggcttgcg ggtcatgcac
     1561 caggtgcgcg gtccttcggg cacctcgacg tcggcggtga cggtgaagcc gagccgctcg
     1621 tagaagggga ggttgcgggg cgcggaggtc tccaggaagg cgggcacccc ggcgcgctcg
     1681 gccgcctcca ctccggggag cacgacggcg ctgcccagac ccttgccctg gtggtcgggc
     1741 gagacgccga cggtggccag gaaccacgcg ggctccttgg gccggtgcgg cgccaggagg
     1801 ccttccatct gttgctgcgc ggccagccgg gaaccgctca actcggccat gcgcgggccg
     1861 atctcggcga acaccgcccc cgcttcgacg ctctccggcg tggtccagac cgccaccgcg
     1921 gcgccgtcgt ccgcgaccca caccttgccg atgtcgagcc cgacgcgcgt gaggaagagt
     1981 tcttgcagct cggtgacccg ctcgatgtgg cggtccggat cgacggtgtg gcgcgtggcg
     2041 gggtagtcgg cgaacgcggc ggcgagggtg cgtacggccc tggggacgtc gtcgcgggtg
     2101 gcgaggcgca ccgtgggctt gtactcggtc atattggctg cagggtcgct cggtgttcga
     2161 ggccacacgc gtcaccttaa tatgcgaagt ggacctggga ccgcgccgcc ccgactgcat
     2221 ctgcgtgttc gaattcgcca atgacaagac gctgggcggg gtttgctcga cattgggtgg
     2281 aaacattcca ggcctgggtg gagaggcttt ttgcttcctc ttgcaaaacc acactgctcg
     2341 actactagtt gattatcttt aacgtacgtc acaatatgat atcccaatac gcgtcaattc
     2401 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     2461 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     2521 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
     2581 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
     2641 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     2701 ttgtctgctc ccggcatccg cttacagaca agctgtgaca gtctccggga gctgcatgtg
     2761 tcagaggttt tcaccgtcat caccgaaacg cgcgagacaa aagggcctcg tgatacgcct
     2821 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     2881 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2941 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3001 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3061 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3121 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3181 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     3241 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3301 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3361 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3421 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3481 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     3541 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3601 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3661 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg gttctcgcgg
     3721 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3781 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3841 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3901 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3961 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4021 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaac
//
