LOCUS       sequence-453955-        2860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Accessory plasmid with a sgRNA cassette for Cas9, and ampicillin 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2860)
  AUTHORS   Xie F, Zhao H, Liu J, Yang X, Neuber M, Agrawal AA, Kaur A, Herrmann
            J, Kalinina OV, Wei X, Muller R, Fu C
  TITLE     Autologous DNA mobilization and multiplication expedite natural 
            products discovery from bacteria.
  JOURNAL   Science. 2024 Dec 13;386(6727):eabq7333. doi: 
            10.1126/science.abq7333. Epub 2024 Dec 13.
  PUBMED    39666857
REFERENCE   2  (bases 1 to 2860)
  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"; journalName: "Science."; 
            date: "2024-12-13"; volume: "386(6727):eabq7333. doi"; pages: " 
            10.1126/science.abq7333. Epub 2024 Dec 13"
COMMENT     Sequence Label: pHelp
FEATURES             Location/Qualifiers
     source          1..2860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        466..541
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(639..655)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(639..655)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(652..674)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    663..679
                     /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(687..693,694..711,712..717))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    732..753
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(870..887)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1041..1629)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1121..1140)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1800..2591,2592..2660))
                     /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     2423..2442
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2661..2765)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2833..2851
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgctagcata tgccttgacc
      421 ttgatgaggc ggcgtgagct acaatcaata ctcgattagc catgggtttt aggtctagaa
      481 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      541 cttttttctg cagctcggta ccaaattcca gaaaagaggc ctcccgaaag gggggccttt
      601 tttcgttttg gtccactagt aagcttggcg taatcatggt catagctgtt tcctgtgtga
      661 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
      721 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
      781 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
      841 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
      901 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
      961 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     1021 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     1081 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1141 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     1201 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     1261 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     1321 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     1381 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     1441 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     1501 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     1561 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1621 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     1681 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     1741 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     1801 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     1861 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     1921 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     1981 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     2041 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     2101 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     2161 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     2221 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     2281 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     2341 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     2401 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     2461 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     2521 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     2581 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     2641 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     2701 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     2761 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     2821 ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
