LOCUS       sequence-365695-        2991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Sub-array backbone 3/4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2991)
  AUTHORS   Shaw WM, Studena L, Roy K, Hapeta P, McCarty NS, Graham AE, Ellis T,
            Ledesma-Amaro R
  TITLE     Inducible expression of large gRNA arrays for multiplexed CRISPRai 
            applications.
  JOURNAL   Nat Commun. 2022 Aug 25;13(1):4984. doi: 10.1038/s41467-022-32603-7.
  PUBMED    36008396
REFERENCE   2  (bases 1 to 2991)
  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: "Nat 
            Commun."; date: "2022-08-25"; volume: "13(1):4984. doi"; pages: " 
            10.1038/s41467-022-32603-7"
COMMENT     Sequence Label: pWS3867
FEATURES             Location/Qualifiers
     source          1..2991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    39..58
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             214..225
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             232..945
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      957..1028
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1044..1071
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(join(1236..2027,2028..2096))
                     /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     1859..1878
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2097..2201)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2304..2892
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2793..2812
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcggccgccc ctgaattcgc atctagacgt ctctctaagt tcactgccgt ataggcagtg
       61 agaccgaaag tgaaacgtga tttcatgcgt cattttgaac attttgtaaa tcttatttaa
      121 taatgtgtgc ggcaattcac atttaattta tgaatgtttt cttaacatcg cggcaactca
      181 agaaacggca ggttcggatc ttagctacta gagaaagagg agaaatacta gatgcgtaaa
      241 ggcgaagagc tgttcactgg tgtcgtccct attctggtgg aactggatgg tgatgtcaac
      301 ggtcataagt tttccgtgcg tggcgagggt gaaggtgacg caactaatgg taaactgacg
      361 ctgaagttca tctgtactac tggtaaactg ccggttcctt ggccgactct ggtaacgacg
      421 ctgacttatg gtgttcagtg ctttgctcgt tatccggacc atatgaagca gcatgacttc
      481 ttcaagtccg ccatgccgga aggctatgtg caggaacgca cgatttcctt taaggatgac
      541 ggcacgtaca aaacgcgtgc ggaagtgaaa tttgaaggcg ataccctggt aaaccgcatt
      601 gagctgaaag gcattgactt taaagaggac ggcaatatcc tgggccataa gctggaatac
      661 aattttaaca gccacaatgt ttacatcacc gccgataaac aaaaaaatgg cattaaagcg
      721 aattttaaaa ttcgccacaa cgtggaggat ggcagcgtgc agctggctga tcactaccag
      781 caaaacactc caatcggtga tggtcctgtt ctgctgccag acaatcacta tctgagcacg
      841 caaagcgttc tgtctaaaga tccgaacgag aaacgcgatc atatggttct gctggagttc
      901 gtaaccgcag cgggcatcac gcatggtatg gatgaactgt acaaatgacc aggcatcaaa
      961 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
     1021 acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttataggt
     1081 ctcatccccg tcagtgacgg agaatgaaga gacgactagt gcactgcagt acagcggccg
     1141 cgattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     1201 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     1261 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     1321 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgggac
     1381 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1441 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1501 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     1561 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     1621 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1681 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     1741 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     1801 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     1861 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     1921 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     1981 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     2041 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     2101 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     2161 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     2221 ccacctgtca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2281 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2341 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2401 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     2461 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2521 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2581 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2641 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2701 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2761 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     2821 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     2881 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     2941 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg t
//
