LOCUS       sequence-249039-        3634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector for assembling gRNA fragments in the second round
            of assembly..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3634)
  AUTHORS   McCarty NS, Shaw WM, Ellis T, Ledesma-Amaro R
  TITLE     Rapid Assembly of gRNA Arrays via Modular Cloning in Yeast.
  JOURNAL   ACS Synth Biol. 2019 Apr 2. doi: 10.1021/acssynbio.9b00041.
  PUBMED    30939239
REFERENCE   2  (bases 1 to 3634)
  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: "ACS Synth 
            Biol. 2019 Apr 2. doi: 10.1021/acssynbio.9b00041."
COMMENT     Sequence Label: Destination Vector
FEATURES             Location/Qualifiers
     source          1..3634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        29..695
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     565..588
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     RBS             859..870
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             877..1590
                     /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      1602..1673
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1689..1716
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    1734..1753
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     terminator      complement(2001..2035)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(2106..2765)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2680..2699
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      2947..3535
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3436..3455
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agtcggtctc aaacgcagtt cgagtttatc attatcaata ctgccatttc aaagaatacg
       61 taaataatta atagtagtga ttttcctaac tttatttagt caaaaaatta gccttttaat
      121 tctgctgtaa cccgtacatg cccaaaatag ggggcgggtt acacagaata tataacatcg
      181 taggtgtctg ggtgaacagt ttattcctgg catccactaa atataatgga gcccgctttt
      241 taagctggca tccagaaaaa aaaagaatcc cagcaccaaa atattgtttt cttcaccaac
      301 catcagttca taggtccatt ctcttagcgc aactacagag aacaggggca caaacaggca
      361 aaaaacgggc acaacctcaa tggagtgatg caacctgcct ggagtaaatg atgacacaag
      421 gcaattgacc cacgcatgta tctatctcat tttcttacac cttctattac cttctgctct
      481 ctctgatttg gaaaaagctg aaaaaaaagg ttgaaaccag ttccctgaaa ttattcccct
      541 acttgactaa taagtatata aagacggtag gtattgattg taattctgta aatctatttc
      601 ttaaacttct taaattctac ttttatagtt agtctttttt ttagttttaa aacaccaaga
      661 acttagtttc gaataaacac acataaacaa acaaaagatc tgccgagacg gaaagtgaaa
      721 cgtgatttca tgcgtcattt tgaacatttt gtaaatctta tttaataatg tgtgcggcaa
      781 ttcacattta atttatgaat gttttcttaa catcgcggca actcaagaaa cggcaggttc
      841 ggatcttagc tactagagaa agaggagaaa tactagatgc gtaaaggcga agagctgttc
      901 actggtgtcg tccctattct ggtggaactg gatggtgatg tcaacggtca taagttttcc
      961 gtgcgtggcg agggtgaagg tgacgcaact aatggtaaac tgacgctgaa gttcatctgt
     1021 actactggta aactgccggt tccttggccg actctggtaa cgacgctgac ttatggtgtt
     1081 cagtgctttg ctcgttatcc ggaccatatg aagcagcatg acttcttcaa gtccgccatg
     1141 ccggaaggct atgtgcagga acgcacgatt tcctttaagg atgacggcac gtacaaaacg
     1201 cgtgcggaag tgaaatttga aggcgatacc ctggtaaacc gcattgagct gaaaggcatt
     1261 gactttaaag aggacggcaa tatcctgggc cataagctgg aatacaattt taacagccac
     1321 aatgtttaca tcaccgccga taaacaaaaa aatggcatta aagcgaattt taaaattcgc
     1381 cacaacgtgg aggatggcag cgtgcagctg gctgatcact accagcaaaa cactccaatc
     1441 ggtgatggtc ctgttctgct gccagacaat cactatctga gcacgcaaag cgttctgtct
     1501 aaagatccga acgagaaacg cgatcatatg gttctgctgg agttcgtaac cgcagcgggc
     1561 atcacgcatg gtatggatga actgtacaaa tgaccaggca tcaaataaaa cgaaaggctc
     1621 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga
     1681 gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacgtctcta tccgttcact
     1741 gccgtatagg cagctcgaga taaagcaatc ttgatgagga taatgatttt tttttgaata
     1801 tacataaata ctaccgtttt tctgctagat tttgtgatga cgtaaataag tacatattac
     1861 tttttaagcc aagacaagat taagcattaa ctttaccctt ttctttctaa gtttcaatat
     1921 tagttatcac tgtttaaaag ttatggcgag aacgtcggcg gttaaaatat attaccctga
     1981 acggctgtga gaccagacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat
     2041 ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc gagctcgata
     2101 tcaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg
     2161 ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc
     2221 ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata
     2281 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga aacgaaaaac
     2341 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct
     2401 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa
     2461 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc
     2521 agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag gcgggcaaga
     2581 atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc
     2641 gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca
     2701 aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc
     2761 tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt
     2821 gatcttattt cattatggtg aaagttggaa cctcttacgt gcccgatcaa tcatgaccaa
     2881 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2941 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3001 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3061 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     3121 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3181 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3241 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3301 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3361 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3421 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3481 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3541 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3601 tcctgcgtta tcccctgatt ctgtggataa ccgt
//
