LOCUS       sequence-405474-        2316 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of sgRNAs in a PlayBack Vector with Kpn1 as a 
            PlayBack Restriction Enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2316)
  AUTHORS   Foran G, Hallam RD, Megaly M, Turgambayeva A, Necakov A
  TITLE     PlayBack cloning: simple, reversible, cost-effective cloning for the
            combinatorial assembly of complex expression constructs.
  JOURNAL   Biotechniques. 2023 Oct;75(4):168-178. doi: 10.2144/btn-2023-0042. 
            Epub 2023 Oct 10.
  PUBMED    37815818
REFERENCE   2  (bases 1 to 2316)
  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: 
            "10.2144/btn-2023-0042"; journalName: "Biotechniques"; date: 
            "2023-10"; volume: "75"; issue: "4"; pages: "168-178"
COMMENT     Sequence Label: PlayBack-U6-Kpn1
FEATURES             Location/Qualifiers
     source          1..2316
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(785..860)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(885..1133)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(943..962)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1113..1133)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        1181..1285
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1286..1354,1355..2146)
                     /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(1504..1523)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatcta ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtat ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagccat ggtcgacaat aggtaccaaa
      781 aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt
      841 gctatttcta gctctaaaac atgtcttcta tatatgaaga ctacggtgtt tcgtcctttc
      901 cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag catatgatag
      961 tccattttaa aacataattt taaaactgca aactacccaa gaaattatta ctttctacgt
     1021 cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattccaat tatctctcta
     1081 acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc ctcggtgaag
     1141 ggtacctata ctcgagttac gcgtaattcg gggaaatgtg cgcggaaccc ctatttgttt
     1201 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     1261 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     1321 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     1381 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     1441 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     1501 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     1561 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     1621 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     1681 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     1741 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     1801 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     1861 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     1921 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     1981 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     2041 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     2101 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     2161 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     2221 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     2281 agcgtcagac cccgtagaaa agatcaaagg atcttc
//
