LOCUS       sequence-397567-        2893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of sgRNAs in a PlayBack Vector with XbaI as a 
            PlayBack Restriction Enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2893)
  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 2893)
  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-Xba1
FEATURES             Location/Qualifiers
     source          1..2893
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(5..109)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     177..195
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(233..255)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     355..374
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        480..728
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     480..500
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     651..670
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        753..828
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(876..892)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(876..892)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(889..911)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    900..916
                     /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(924..930,931..948,949..954))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    969..990
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1107..1124)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1278..1866)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1358..1377)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2037..2828,2829..2893,1..4))
                     /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     2660..2679
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
       61 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
      121 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
      181 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
      241 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
      301 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
      361 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
      421 ggagaaaata ccgcatcagg cgccaattac gcgtaactcg agtatatcta gacttcaccg
      481 agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga
      541 taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa
      601 agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata
      661 tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac
      721 gaaacaccgt agtcttcata tatagaagac atgttttaga gctagaaata gcaagttaaa
      781 ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttttctag
      841 atattgtcga ccatggcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
      901 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
      961 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     1021 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     1081 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1141 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     1201 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1261 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1321 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1381 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     1441 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     1501 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     1561 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     1621 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     1681 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     1741 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     1801 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     1861 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     1921 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     1981 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2041 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2101 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2161 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     2221 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2281 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     2341 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     2401 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     2461 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     2521 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     2581 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     2641 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     2701 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     2761 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     2821 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     2881 aaatgttgaa tac
//
