LOCUS       sequence-197186-        3906 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA entry vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3906)
  TITLE     Ellis Lab Yeast CRISPR Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 3906)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pWS082
FEATURES             Location/Qualifiers
     source          1..3906
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     116..135
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             985..996
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1003..1716
                     /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      1728..1799
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1815..1842
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        1856..1931
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(join(2465..3256,3257..3325))
                     /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     3088..3107
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3326..3430)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(3533..3906,1..215)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
       61 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
      121 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
      181 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
      241 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
      301 ctgtggataa ccgtgcggcc gcccctgata tcaacaacag aggacatatg ccctacctcc
      361 atgatctact gacgtccctg aggctgcaat tcatgtaatg gggcagtatc cgcggcaagt
      421 cctagtgcaa tggcggtttt ttaccctcgt tctgaagaag aggcgacgcg ggtgcggtca
      481 tcactaatgt ggaaattggt aagactctcg ggcctccgcc tttaggcggt gcttactctt
      541 tcataaaggg gctgttagtt atgcaacgat gtgcttcagt attacatttt ttgccttcaa
      601 cgccttgatt gttctatttt tgctaataat aaatctattt catcggacta aaagtccatt
      661 agttgtaagc ggatttagct cagttgggag agcgccagac tgaagaaaaa cttcggtcaa
      721 gtcatctgga ggtcctgtgt tcgatccaca gaattcgcag atggccggca tggtcccagc
      781 ctcctcgctg gcgccggctg ggcaacacct tcgggtggcg aatgggactt gagacggaaa
      841 gtgaaacgtg atttcatgcg tcattttgaa cattttgtaa atcttattta ataatgtgtg
      901 cggcaattca catttaattt atgaatgttt tcttaacatc gcggcaactc aagaaacggc
      961 aggttcggat cttagctact agagaaagag gagaaatact agatgcgtaa aggcgaagag
     1021 ctgttcactg gtgtcgtccc tattctggtg gaactggatg gtgatgtcaa cggtcataag
     1081 ttttccgtgc gtggcgaggg tgaaggtgac gcaactaatg gtaaactgac gctgaagttc
     1141 atctgtacta ctggtaaact gccggttcct tggccgactc tggtaacgac gctgacttat
     1201 ggtgttcagt gctttgctcg ttatccggac catatgaagc agcatgactt cttcaagtcc
     1261 gccatgccgg aaggctatgt gcaggaacgc acgatttcct ttaaggatga cggcacgtac
     1321 aaaacgcgtg cggaagtgaa atttgaaggc gataccctgg taaaccgcat tgagctgaaa
     1381 ggcattgact ttaaagagga cggcaatatc ctgggccata agctggaata caattttaac
     1441 agccacaatg tttacatcac cgccgataaa caaaaaaatg gcattaaagc gaattttaaa
     1501 attcgccaca acgtggagga tggcagcgtg cagctggctg atcactacca gcaaaacact
     1561 ccaatcggtg atggtcctgt tctgctgcca gacaatcact atctgagcac gcaaagcgtt
     1621 ctgtctaaag atccgaacga gaaacgcgat catatggttc tgctggagtt cgtaaccgca
     1681 gcgggcatca cgcatggtat ggatgaactg tacaaatgac caggcatcaa ataaaacgaa
     1741 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct
     1801 actagagtca cactggctca ccttcgggtg ggcctttctg cgtttatacg tctcagtttt
     1861 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     1921 cgagtcggtg ctttttttat tttttgtcac tattgttatg taaaatgcca cctctgacag
     1981 tatggaacgc aaacttctgt ctagtggata gctggacatt ccaggcagtg cctctgccgc
     2041 cggacccctc tcgtgattgg gtagctggac atgcccttgt aagatataac aagagcctgc
     2101 ctgtctaatg atctcacggc gaaagtcggg gagacagcag cggctgcaga cattataccg
     2161 caacaacact aaggtgagat aactccgtac attgactacg cgttcctcta gaccttactt
     2221 gaccggatac agtgtctttg acacgtttat gggttacagc aatcacatcc aagactggct
     2281 atgcacgaag caactcttga gtgttaaaat gttgacccct gtatttggga tgcgggtagt
     2341 agatgagtgc aggatatcta cagcggccgc gattatcaaa aaggatcttc acctagatcc
     2401 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2461 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2521 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2581 gccccagtgc tgcaatgata ccgcgggacc cacgctcacc ggctccagat ttatcagcaa
     2641 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2701 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2761 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2821 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2881 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2941 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3001 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3061 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3121 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3181 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3241 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3301 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3361 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3421 gggttccgcg cacatttccc cgaaaagtgc cacctgtcat gaccaaaatc ccttaacgtg
     3481 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3541 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     3601 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     3661 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     3721 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     3781 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3841 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3901 aactga
//
