LOCUS       sequence-397572-        2897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of sgRNAs in a PlayBack Vector with NotI as a 
            PlayBack Restriction Enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2897)
  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 2897)
  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-Not1
FEATURES             Location/Qualifiers
     source          1..2897
                     /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"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        complement(1041..1116)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1141..1389)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1199..1218)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1369..1389)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1497..1516)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1616..1638
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1676..1694)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1762..1866
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1867..1935,1936..2727)
                     /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(2085..2104)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg 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 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg ccatggtcga
     1021 caatagcggc cgcaaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact
     1081 agccttattt taacttgcta tttctagctc taaaacatgt cttctatata tgaagactac
     1141 ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac
     1201 ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa
     1261 ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat
     1321 tccaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa
     1381 taggccctcg gtgaaggcgg ccgctatact cgagttacgc gtaattggcg cctgatgcgg
     1441 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     1501 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     1561 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     1621 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     1681 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     1741 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     1801 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     1861 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     1921 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     1981 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     2041 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     2101 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     2161 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     2221 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     2281 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     2341 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     2401 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     2461 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     2521 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     2581 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     2641 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     2701 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     2761 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     2821 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2881 aagatcaaag gatcttc
//
