LOCUS       sequence-397393-        2893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of sgRNAs in a PlayBack Vector with Bamh1 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-Bamh1
FEATURES             Location/Qualifiers
     source          1..2893
                     /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(1039..1114)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1139..1387)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1197..1216)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1367..1387)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1493..1512)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1612..1634
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1672..1690)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1758..1862
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1863..1931,1932..2723)
                     /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(2081..2100)
                     /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 caataggatc caaaaaaagc accgactcgg tgccactttt tcaagttgat aacggactag
     1081 ccttatttta acttgctatt tctagctcta aaacatgtct tctatatatg aagactacgg
     1141 tgtttcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg
     1201 taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt
     1261 attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc
     1321 caattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata
     1381 ggccctcggt gaagggatcc tatactcgag ttacgcgtaa ttggcgcctg atgcggtatt
     1441 ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct
     1501 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     1561 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     1621 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     1681 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     1741 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     1801 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     1861 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     1921 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     1981 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     2041 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     2101 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     2161 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     2221 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     2281 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     2341 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     2401 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     2461 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     2521 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     2581 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     2641 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     2701 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     2761 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     2821 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2881 tcaaaggatc ttc
//
