LOCUS       sequence-250070-        3075 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the spCas9 gRNA GFP dropout expression cassette (bovine u6 
            promoter and constant region 2) 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 3075)
  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 3075)
  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_055
FEATURES             Location/Qualifiers
     source          1..3075
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     415..434
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             1073..1084
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1091..1804
                     /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      1816..1887
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1903..1930
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2055..2089)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(2160..2819)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2734..2753
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      join(3001..3075,1..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 ctcaaacgcg tgaccgagct tgtctgcccg cgcagtccac
      661 tagacagacg cgcagaggtc gggcgggcct agggccaatt tcccatggtt cccttcattt
      721 gcatatatga tgtaataagg ttatggagac tattaaactt agccctaatc aaactatatg
      781 atgataatgt gtgtggtaca aaaggtcata acttattata tactttgaaa cttaaaaaag
      841 ggttacagtt tagtcaccat aactgtaaaa ttttttctat tcttagcttt atatagttct
      901 tgagaggcca tgtttatgga gacggaaagt gaaacgtgat ttcatgcgtc attttgaaca
      961 ttttgtaaat cttatttaat aatgtgtgcg gcaattcaca tttaatttat gaatgttttc
     1021 ttaacatcgc ggcaactcaa gaaacggcag gttcggatct tagctactag agaaagagga
     1081 gaaatactag atgcgtaaag gcgaagagct gttcactggt gtcgtcccta ttctggtgga
     1141 actggatggt gatgtcaacg gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc
     1201 aactaatggt aaactgacgc tgaagttcat ctgtactact ggtaaactgc cggttccttg
     1261 gccgactctg gtaacgacgc tgacttatgg tgttcagtgc tttgctcgtt atccggacca
     1321 tatgaagcag catgacttct tcaagtccgc catgccggaa ggctatgtgc aggaacgcac
     1381 gatttccttt aaggatgacg gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga
     1441 taccctggta aaccgcattg agctgaaagg cattgacttt aaagaggacg gcaatatcct
     1501 gggccataag ctggaataca attttaacag ccacaatgtt tacatcaccg ccgataaaca
     1561 aaaaaatggc attaaagcga attttaaaat tcgccacaac gtggaggatg gcagcgtgca
     1621 gctggctgat cactaccagc aaaacactcc aatcggtgat ggtcctgttc tgctgccaga
     1681 caatcactat ctgagcacgc aaagcgttct gtctaaagat ccgaacgaga aacgcgatca
     1741 tatggttctg ctggagttcg taaccgcagc gggcatcacg catggtatgg atgaactgta
     1801 caaatgacca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     1861 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     1921 cctttctgcg tttatacgtc tcgtttgaga gctaagcaga aagctgcata gcaagttcaa
     1981 ataaggctag tccgtacaca acttgaaaaa gtggcagccg agtcggctgc tttttttgct
     2041 gtgagaccag accaataaaa aacgcccggc ggcaaccgag cgttctgaac aaatccagat
     2101 ggagttctga ggtcattact ggatctatca acaggagtcc aagcgagctc gatatcaaat
     2161 tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca
     2221 tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg
     2281 ccttgcgtat aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc
     2341 acgtttaaat caaaactggt gaaactcacc cagggattgg ctgaaacgaa aaacatattc
     2401 tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa
     2461 tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt
     2521 tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca
     2581 ccgtctttca ttgccatacg aaattccgga tgagcattca tcaggcgggc aagaatgtga
     2641 ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata
     2701 tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt
     2761 tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt
     2821 ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt
     2881 atttcattat ggtgaaagtt ggaacctctt acgtgcccga tcaatcatga ccaaaatccc
     2941 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3001 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3061 agcggtggtt tgttt
//
