LOCUS       sequence-250066-        3087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the saCas9 gRNA GFP dropout expression cassette as a type 
            234 part to be used in the MTK system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3087)
  AUTHORS   Fonseca JP, Bonny AR, Kumar GR, Ng AH, Town J, Wu QC, Aslankoohi E, 
            Chen SY, Dods G, Harrigan P, Osimiri LC, Kistler AL, El-Samad H
  TITLE     A Toolkit for Rapid Modular Construction of Biological Circuits in 
            Mammalian Cells.
  JOURNAL   ACS Synth Biol. 2019 Nov 15;8(11):2593-2606. doi: 
            10.1021/acssynbio.9b00322. Epub 2019 Nov 5.
  PUBMED    31686495
REFERENCE   2  (bases 1 to 3087)
  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: 
            "10.1021/acssynbio.9b00322"; journalName: "ACS Synth Biol"; date: 
            "2019-11-15- 15"; volume: "8"; issue: "11"; pages: "2593-2606"
COMMENT     Sequence Label: MTK234_050
FEATURES             Location/Qualifiers
     source          1..3087
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     415..434
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(823..856)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     860..880
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     RBS             1097..1108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1115..1828
                     /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      1840..1911
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1927..1954
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        1967..2042
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     terminator      complement(2067..2101)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(2172..2831)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2746..2765
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      join(3013..3087,1..514)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
       61 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
      121 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
      181 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
      241 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
      301 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
      361 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
      421 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
      481 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
      541 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
      601 tgtggataac cgtagtcggt ctcaaacgga tccgacgcgc catctctagg cccgcgccgg
      661 ccccctcgca cggacttgtg ggagaagctc ggctactccc ctgccccggt taatttgcat
      721 ataatatttc ctagtaacta tagaggctta atgtgcgata aaagacagat aatctgttct
      781 ttttaatact agctacattt tacatgatag gcttggattt ctataacttc gtatagcata
      841 cattatacga agttataaac agcacaaaag gaaactcacc ctaactgtaa agtaattgtg
      901 tgttttgaga ctataagtat cccttggaga accaccttgt tggagacgga aagtgaaacg
      961 tgatttcatg cgtcattttg aacattttgt aaatcttatt taataatgtg tgcggcaatt
     1021 cacatttaat ttatgaatgt tttcttaaca tcgcggcaac tcaagaaacg gcaggttcgg
     1081 atcttagcta ctagagaaag aggagaaata ctagatgcgt aaaggcgaag agctgttcac
     1141 tggtgtcgtc cctattctgg tggaactgga tggtgatgtc aacggtcata agttttccgt
     1201 gcgtggcgag ggtgaaggtg acgcaactaa tggtaaactg acgctgaagt tcatctgtac
     1261 tactggtaaa ctgccggttc cttggccgac tctggtaacg acgctgactt atggtgttca
     1321 gtgctttgct cgttatccgg accatatgaa gcagcatgac ttcttcaagt ccgccatgcc
     1381 ggaaggctat gtgcaggaac gcacgatttc ctttaaggat gacggcacgt acaaaacgcg
     1441 tgcggaagtg aaatttgaag gcgataccct ggtaaaccgc attgagctga aaggcattga
     1501 ctttaaagag gacggcaata tcctgggcca taagctggaa tacaatttta acagccacaa
     1561 tgtttacatc accgccgata aacaaaaaaa tggcattaaa gcgaatttta aaattcgcca
     1621 caacgtggag gatggcagcg tgcagctggc tgatcactac cagcaaaaca ctccaatcgg
     1681 tgatggtcct gttctgctgc cagacaatca ctatctgagc acgcaaagcg ttctgtctaa
     1741 agatccgaac gagaaacgcg atcatatggt tctgctggag ttcgtaaccg cagcgggcat
     1801 cacgcatggt atggatgaac tgtacaaatg accaggcatc aaataaaacg aaaggctcag
     1861 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct ctactagagt
     1921 cacactggct caccttcggg tgggcctttc tgcgtttata cgtctcgtta tagtactctg
     1981 gaaacagaat ctactataac aaggcaaaat gccgtgttta tctcgtcaac ttgttggcga
     2041 gatttttttg ctgtgagacc agaccaataa aaaacgcccg gcggcaaccg agcgttctga
     2101 acaaatccag atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc
     2161 tcgatatcaa attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc
     2221 attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc
     2281 agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg
     2341 tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgaaacg
     2401 aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc
     2461 acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc
     2521 gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat
     2581 atcaccagct caccgtcttt cattgccata cgaaattccg gatgagcatt catcaggcgg
     2641 gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa
     2701 aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat
     2761 gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt
     2821 tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa aaatacgccc
     2881 ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccc gatcaatcat
     2941 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     3001 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     3061 accaccgcta ccagcggtgg tttgttt
//
