LOCUS       sequence-250078-        3672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the spCas9 gRNA GFP dropout expression cassette with ConLS 
            and ConRE connectors with Ampicillin resistance as a type 0 part to 
            be used in the MTK system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3672)
  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 3672)
  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: MTK0_001
FEATURES             Location/Qualifiers
     source          1..3672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     55..74
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(293..397)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      500..1088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     989..1008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(1588..1621)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1625..1645
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     RBS             1862..1873
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1880..2593
                     /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      2605..2676
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2692..2719
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(join(3104..3672,1..223,224..292))
                     /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"
ORIGIN
        1 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
       61 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
      121 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
      181 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
      241 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
      301 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
      361 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
      421 ctgtcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
      481 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
      541 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
      601 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
      661 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
      721 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
      781 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
      841 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
      901 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
      961 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1021 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1081 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1141 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtgcg gccgcccctg
     1201 aattcgcatc tagaacgggg tcatcacggc tcatcatgcg ccaaacaaat gtgtgcaata
     1261 cacgctcgga tgactgcatg atgaccgcac tgactgggga cagcagatcc acctaagcct
     1321 gtgagagaag cagacacccg acagatcaag gcagttacgt ctcaccaaac cagatgtcaa
     1381 cacagctaca acggatccga cgcgccatct ctaggcccgc gccggccccc tcgcacggac
     1441 ttgtgggaga agctcggcta ctcccctgcc ccggttaatt tgcatataat atttcctagt
     1501 aactatagag gcttaatgtg cgataaaaga cagataatct gttcttttta atactagcta
     1561 cattttacat gataggcttg gatttctata acttcgtata gcatacatta tacgaagtta
     1621 taaacagcac aaaaggaaac tcaccctaac tgtaaagtaa ttgtgtgttt tgagactata
     1681 agtatccctt ggagaaccac cttgttggag acggaaagtg aaacgtgatt tcatgcgtca
     1741 ttttgaacat tttgtaaatc ttatttaata atgtgtgcgg caattcacat ttaatttatg
     1801 aatgttttct taacatcgcg gcaactcaag aaacggcagg ttcggatctt agctactaga
     1861 gaaagaggag aaatactaga tgcgtaaagg cgaagagctg ttcactggtg tcgtccctat
     1921 tctggtggaa ctggatggtg atgtcaacgg tcataagttt tccgtgcgtg gcgagggtga
     1981 aggtgacgca actaatggta aactgacgct gaagttcatc tgtactactg gtaaactgcc
     2041 ggttccttgg ccgactctgg taacgacgct gacttatggt gttcagtgct ttgctcgtta
     2101 tccggaccat atgaagcagc atgacttctt caagtccgcc atgccggaag gctatgtgca
     2161 ggaacgcacg atttccttta aggatgacgg cacgtacaaa acgcgtgcgg aagtgaaatt
     2221 tgaaggcgat accctggtaa accgcattga gctgaaaggc attgacttta aagaggacgg
     2281 caatatcctg ggccataagc tggaatacaa ttttaacagc cacaatgttt acatcaccgc
     2341 cgataaacaa aaaaatggca ttaaagcgaa ttttaaaatt cgccacaacg tggaggatgg
     2401 cagcgtgcag ctggctgatc actaccagca aaacactcca atcggtgatg gtcctgttct
     2461 gctgccagac aatcactatc tgagcacgca aagcgttctg tctaaagatc cgaacgagaa
     2521 acgcgatcat atggttctgc tggagttcgt aaccgcagcg ggcatcacgc atggtatgga
     2581 tgaactgtac aaatgaccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     2641 tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct
     2701 tcgggtgggc ctttctgcgt ttatacgtct cgtttaagag ctaagctgga aacagcatag
     2761 caagtttaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     2821 ttttgctgag catgagacgg aaatctgctc gtcagtggtg ctcacactga cgaatcatgt
     2881 acagatcata ccgatgactg cctggcgact cacaactaag caagacagcc ggaaccagcg
     2941 ccggcgaaca ccactgcata tatggcatat cacaacagtc caactagtgc actgcagtac
     3001 agcggccgcg attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3061 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3121 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3181 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3241 cgcgggaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3301 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     3361 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     3421 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3481 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3541 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3601 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3661 caaccaagtc at
//
