LOCUS       sequence-330265-        3954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pgRNA_CV2 is derived from gRNA_cloning vector (Addgene plasmid ID: 
            41824) by adding about 80 bps from the sgRNA sequence as well as an 
            AgeI site. In the literature, sgRNA_AL is used as an alias..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3954)
  AUTHORS   Sheng C, Mendler IH, Rieke S, Snyder P, Jentsch M, Friedrich D, 
            Drossel B, Loewer A
  TITLE     PCNA-Mediated Degradation of p21 Coordinates the DNA Damage Response
            and Cell Cycle Regulation in Individual Cells.
  JOURNAL   Cell Rep. 2019 Apr 2;27(1):48-58.e7. doi: 
            10.1016/j.celrep.2019.03.031.
  PUBMED    30943414
REFERENCE   2  (bases 1 to 3954)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2019.03"; journalName: "Cell Rep"; date: 
            "2019-04-2- 2"; volume: "27"; issue: "1"; pages: "48-58.e7"
COMMENT     Sequence Label: pgRNA_CV2
FEATURES             Location/Qualifiers
     source          1..3954
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(85..104)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     408..430
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     422..439
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     423..439
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     446..465
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        446..464
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        604..852
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     604..624
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     775..794
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        854..929
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(941..965)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(1050..1068)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1051..1068)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1086..1102)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1086..1102)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1099..1121)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1110..1126
                     /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)."
     promoter        complement(join(1134..1140,1141..1158,1159..1164))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1179..1200
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1317..1334)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1488..2076)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1568..1587)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2214..2588)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     regulatory      2585..2594
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(2794..3588)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(2794..3588)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2905..2924)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3515..3534
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     CDS             complement(join(3937..3954,1..285))
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
ORIGIN
        1 caggttaatg gcgtttttga tgtcattttc gcggtggctg agatcagcca cttcttcccc
       61 gataacggag accggcacac tggccatatc ggtggtcatc atgcgccagc tttcatcccc
      121 gatatgcacc accgggtaaa gttcacggga gactttatct gacagcagac gtgcactggc
      181 cagggggatc accatccgtc gccccggcgt gtcaataata tcactctgta catccacaaa
      241 cagacgataa cggctctctc ttttataggt gtaaacctta aactgccgta cgtataggct
      301 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      361 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      421 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggccctct
      481 agatgcatgc tcgagcggcc gccagtgtga tggatatctg cagaattcgc cctttgtaca
      541 aaaaagcagg ctttaaagga accaattcag tcgactggat ccggtaccaa ggtcgggcag
      601 gaagagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
      661 gagataatta gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
      721 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
      781 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
      841 ggacgaaaca ccggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc
      901 aacttgaaaa agtggcaccg agtcggtgct ttttttctag acccagcttt cttgtacaaa
      961 gttggcatta aagggcgaat tccagcacac tggcggccgt tactagtgga tccgagctcg
     1021 gtaccaagct tgatgcatag cttgagtatt ctatagtgtc acctaaatag cttggcgtaa
     1081 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     1141 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     1201 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     1261 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     1321 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     1381 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     1441 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     1501 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     1561 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1621 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1681 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1741 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1801 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1861 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     1921 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     1981 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2041 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2101 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2161 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttagcac gtgtcagtcc
     2221 tgctcctcgg ccacgaagtg cacgcagttg ccggccgggt cgcgcagggc gaactcccgc
     2281 ccccacggct gctcgccgat ctcggtcatg gccggcccgg aggcgtcccg gaagttcgtg
     2341 gacacgacct ccgaccactc ggcgtacagc tcgtccaggc cgcgcaccca cacccaggcc
     2401 agggtgttgt ccggcaccac ctggtcctgg accgcgctga tgaacagggt cacgtcgtcc
     2461 cggaccacac cggcgaagtc gtcctccacg aagtcccggg agaacccgag ccggtcggtc
     2521 cagaactcga ccgctccggc gacgtcgcgc gcggtgagca ccggaacggc actggtcaac
     2581 ttggccatgg tggccctcct cacgtgctat tattgaagca tttatcaggg ttattgtctc
     2641 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     2701 tttccccgaa aagtgccacc tgatgcggtg tgaaataccg cacagatgcg taaggagaaa
     2761 ataccgcatc aggaaattgt aagcgttaat aattcagaag aactcgtcaa gaaggcgata
     2821 gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc
     2881 ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg
     2941 gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat
     3001 gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgct
     3061 cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc
     3121 atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc
     3181 ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc
     3241 catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac
     3301 ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca
     3361 aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgca gttcattcag
     3421 ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa
     3481 cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc
     3541 cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc
     3601 tcatcctgtc tcttgatcag agcttgatcc cctgcgccat cagatccttg gcggcaagaa
     3661 agccatccag tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa
     3721 ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg taagcccact
     3781 gcaagctacc tgctttctct ttgcgcttgc gttttccctt gtccagatag cccagtagct
     3841 gacattcatc cggggtcagc accgtttctg cggactggct ttctacgtga aaaggatcta
     3901 ggtgaagatc ctttttgata atctcatgcc tgacatttat attccccaga acat
//
