LOCUS       sequence-250076-        3034 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the spCas9 gRNA GFP dropout expression cassette (human u6 
            promoter and constant region 3) 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 3034)
  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 3034)
  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_056
FEATURES             Location/Qualifiers
     source          1..3034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     415..434
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     629..649
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     800..819
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     RBS             1032..1043
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1050..1763
                     /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
                     TYKTRAEVKFKGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1775..1846
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1862..1889
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2014..2048)
                     /label=lambda t0 terminator
                     /note="minimal transcription terminator from phage lambda 
                     (Scholtissek and Grosse, 1987)"
     CDS             complement(2119..2778)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2693..2712
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      join(2960..3034,1..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 gggcctattt cccatgattc cttcatattt
      661 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
      721 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
      781 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
      841 cttggcttta tatatcttgt ggaaagccag aaacatggag acggaaagtg aaacgtgatt
      901 tcatgcgtca ttttgaacat tttgtaaatc ttatttaata atgtgtgcgg caattcacat
      961 ttaatttatg aatgttttct taacatcgcg gcaactcaag aaacggcggg ttcggatctt
     1021 agctactaga gaaagaggag aaatactaga tgcgtaaagg cgaagagctg ttcactggtg
     1081 tcgtccctat tctggtggaa ctggatggtg atgtcaacgg tcataagttt tccgtgcgtg
     1141 gcgagggtga aggtgacgca actaatggta aactgacgct gaagttcatc tgtactactg
     1201 gtaaactgcc ggttccttgg ccgactctgg taacgacgct gacttatggt gttcagtgct
     1261 ttgctcgtta tccggaccat atgaagcagc atgacttctt caagtccgcc atgccggaag
     1321 gctatgtgca ggaacgcacg atttccttta aggatgacgg cacgtacaaa acgcgtgcgg
     1381 aagtgaaatt taaaggcgat accctggtaa accgcattga gctgaaaggc attgacttta
     1441 aagaggacgg caatatcctg ggccataagc tggaatacaa ttttaacagc cacaatgttt
     1501 acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa ttttaaaatt cgccacaacg
     1561 tggaggatgg cagcgtgcag ctggctgatc actaccagca aaacactcca atcggtgatg
     1621 gtcctgttct gctgccagac aatcactatc tgagcacgca aagcgttctg tctaaagatc
     1681 cgaacgagaa acgcgatcat atggttctgc tggagttcgt aaccgcagcg ggcatcacgc
     1741 atggtatgga tgaactgtac aaatgaccag gcatcaaata aaacgaaagg ctcagtcgaa
     1801 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac
     1861 tggctcacct tcgggtgggc ctttctgcgt ttatacgtct cgtttcagag ctaagcacaa
     1921 gagtgcatag caagttgaaa taaggctagt ccgtttacaa cttgaaaaag tggcacccga
     1981 gtcgggtgct ttttttgctg tgagaccaga ccaataaaaa acgcccggcg gcaaccgagc
     2041 gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca
     2101 agcgagctcg atatcaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc
     2161 attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag
     2221 cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa
     2281 gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc
     2341 tgaaacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta
     2401 acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact
     2461 ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact
     2521 atcccatatc accagctcac cgtctttcat tgccatacga aattccggat gagcattcat
     2581 caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt
     2641 ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga
     2701 ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc
     2761 agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa
     2821 tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgcccgat
     2881 caatcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2941 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3001 caaaaaaacc accgctacca gcggtggttt gttt
//
