LOCUS       sequence-272155-        5986 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the Cas9 cROSA26 homology destination with Blasticidin 
            resistance cassette GFP dropout destination vector 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 5986)
  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 5986)
  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_004
FEATURES             Location/Qualifiers
     source          1..5986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     566..585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(781..797)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(781..797)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     RBS             1522..1533
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1540..2253
                     /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      2265..2336
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2352..2379
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        3507..3710
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3661..3681
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3732..3741
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3738..4136
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(4148..4165)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4154..4361
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(5023..5838)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5023..5838)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5746..5765
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5830..5849
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtgcggcc gccaatcatg
      781 gtcatagctg tttcctgcaa cggaaagggc ctaaaatttt aagattagta tctaatttaa
      841 aatgctacac tttggtggtg taaacaacgt tctcaaacga cgtgagtaaa aaactcagat
      901 gaccatgccc ttaacctcct tttatccaca ggaaaagcac tacaaaaacg atcttaagta
      961 gtaataacac gcagccttgc tgcataaaat cagcccgaga tgaatgcctg gactcccatt
     1021 tccctcctcc acccctatta taattatttt gtgacacaaa ctcaagcagg gtgaagagag
     1081 aaatctcaca cctgctcaat cccctgcagg acaacgccca gacaccaggt aaagcctggg
     1141 cacagagagt cccgcccaca atcgagaagg agcaactcga catggaggcg atgacgccct
     1201 gaattcgcat ctagactgat gagacgtggt agagccacaa acagccggta caagcaacga
     1261 tctccaggac catctgaatc atgcgcggat gacacgaact cacgacggcg atcacagaca
     1321 ttaacccaca gtacagacac tgcgacaacg tggcaattcg tcgcaataca acggaaagtg
     1381 aaacgtgatt tcatgcgtca ttttgaacat tttgtaaatc ttatttaata atgtgtgcgg
     1441 caattcacat ttaatttatg aatgttttct taacatcgcg gcaactcaag aaacggcagg
     1501 ttcggatctt agctactaga gaaagaggag aaatactaga tgcgtaaagg cgaagagctg
     1561 ttcactggtg tcgtccctat tctggtggaa ctggatggtg atgtcaacgg tcataagttt
     1621 tccgtgcgtg gcgagggtga aggtgacgca actaatggta aactgacgct gaagttcatc
     1681 tgtactactg gtaaactgcc ggttccttgg ccgactctgg taacgacgct gacttatggt
     1741 gttcagtgct ttgctcgtta tccggaccat atgaagcagc atgacttctt caagtccgcc
     1801 atgccggaag gctatgtgca ggaacgcacg atttccttta aggatgacgg cacgtacaaa
     1861 acgcgtgcgg aagtgaaatt tgaaggcgat accctggtaa accgcattga gctgaaaggc
     1921 attgacttta aagaggacgg caatatcctg ggccataagc tggaatacaa ttttaacagc
     1981 cacaatgttt acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa ttttaaaatt
     2041 cgccacaacg tggaggatgg cagcgtgcag ctggctgatc actaccagca aaacactcca
     2101 atcggtgatg gtcctgttct gctgccagac aatcactatc tgagcacgca aagcgttctg
     2161 tctaaagatc cgaacgagaa acgcgatcat atggttctgc tggagttcgt aaccgcagcg
     2221 ggcatcacgc atggtatgga tgaactgtac aaatgaccag gcatcaaata aaacgaaagg
     2281 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact
     2341 agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatagctgg aaatctgctc
     2401 gtcagtggtg ctcacactga cgaatcatgt acagatcata ccgatgactg cctggcgact
     2461 cacaactaag caagacagcc ggaaccagcg ccggcgaaca ccactgcata tatggcatat
     2521 cacaacagtc cacgtctcaa gcagttacag agatgttacg aaccactagt gcactgcaga
     2581 tcgaattcga gttggcgcgc ctgtcattct aaatctctct ttcagcctaa agctttttcc
     2641 ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg
     2701 atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg
     2761 gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga
     2821 gggacgtaat tacatccctg ggggctttgg gagggggctg tccccgtgag ctcccaggcg
     2881 cgcctgtcat tctaaatctc tctttcagcc taaagctttt tccccgtatc cccccaggtg
     2941 tctgcaggct caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc
     3001 ccgtgcccgg gctgtccccg cacgctgccg gctcggggat gcggggggag cgccggaccg
     3061 gagcggagcc ccgggcggct cgctgctgcc ccctagcggg ggagggacgt aattacatcc
     3121 ctgggggctt tggggggggg ctgtccccgt gagctcaata aaagagccca ctacagcggt
     3181 gggctctatg gggtacctac gtaggtacat aacttacggt aaatggcccg cctggctgac
     3241 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     3301 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     3361 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     3421 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     3481 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
     3541 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     3601 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     3661 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga
     3721 accgtcagat agccaccatg gccaagcctt tgtctcaaga agaatccacc ctcattgaaa
     3781 gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc gccagcgcag
     3841 ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt actgggggac
     3901 cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc aacctgactt
     3961 gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga cggtgccgac
     4021 aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt gatggacagc
     4081 cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag ggctaactcg
     4141 agcgacgcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     4201 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     4261 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     4321 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgagt
     4381 agatcgcggg ggagggaggg attcttctag gcccagggcg gtccttagga gacaggaggc
     4441 agccgagaac tcccataaag gtattgcggc actcccctcc ccctaccgag aagggtgcgg
     4501 ccttctctcc gcctcctcca ctgcagatcc ctcaggattg cagctcgcgc cgattttcag
     4561 agatcacgca cctccctccc acaaaccagc aggaccgacc cccgctcctc cccccacctc
     4621 cgcgagtccg cgagcggatc cagctcatct cgtccttcag gcggcggacg cccgggacgg
     4681 agccgcggtc gccgccaccc agaagcctcg acccagcagt ccgcctccgc ctccgcctcc
     4741 gcctctagtg cgtgcgcagg ggcgcggggc cagacactgc ggcgcggggc ctcgggaagg
     4801 accgtaccaa cgaccgcctc cctgcccctc cgccccctct gctgacccgc tcagtggttt
     4861 cgctcaattg ggacccgagc caatggcaag gggccgttcc ccggcttcag cagccaatca
     4921 ggtgccgaag ccacacgggc ggcgggtaac cctactcccc gcgagggttt tcccagtcac
     4981 gacccgagcg gccgcgtgtt acaaccaatt aaccaattct gattagaaaa actcatcgag
     5041 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     5101 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     5161 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     5221 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     5281 caaaagctta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     5341 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgaggcgaaa
     5401 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     5461 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     5521 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     5581 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     5641 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     5701 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     5761 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctggagc aagacgtttc
     5821 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     5881 tgttcatgat gatatatttt tatcttgtgc aatgtaacat cagagatttt gagacacaac
     5941 gtggctttgt tgaataaatc gaacttttgc tgagttgaag gatcag
//
