LOCUS       sequence-264211-        6755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the Cas9 hCLYBL homology destination with Hygromycin 
            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 6755)
  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 6755)
  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_047
FEATURES             Location/Qualifiers
     source          1..6755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     146..165
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             1636..1647
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1654..2367
                     /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      2379..2450
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2466..2493
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2979..3078
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     primer_bind     3029..3049
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3100..3109
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3106..4134
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             3106..4134
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     primer_bind     complement(4146..4163)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4152..4359
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(5372..6187)
                     /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(5372..6187)
                     /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     6095..6114
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6179..6198
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(6412..6755,1..245)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
       61 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      121 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      181 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      241 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      301 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtgcggcc gccaatagcc
      361 ttatacctcc tcatctcaaa acttattaca aaaccacagt tttcaaaaca gtgtggtact
      421 gcacatatat agaccaatgg actaggatag agagcccata agtaaaccct tgtatgtatg
      481 gtcagcagga gaatcatgcc aatgggccaa tatacattct gacccacagt ttcataataa
      541 aataaaatgg ttgtggttgt aagccactat gtttcagagt ggtttgttac acagcaataa
      601 ataactaata tagtaggcat accatcaagt ccaaagtagg tagagaagaa tgtaaatagc
      661 agagcaaaac agcatgactg gtggctggga ggcttaaaac tgggacagga tcagagtcat
      721 gaaagaagtc aaagaaatgg ttcagaagta aggctgagac tgacttacaa aagctgaaag
      781 tccctttaag ttggtgtttg gtgcattggc aggggcaggt atggtgactt aaaagagcca
      841 tgctcaacaa gatcaagcac aacacaatca cgggtcaccc cagcagacct tagcgagtct
      901 agccatttct ttggtggtgg tcacagtcat gcttcagccc agtttccact tggacaaatg
      961 gtacatattt tcaatgagat gaaaattaag atacaatcca tgtgctcaga gagtgatcac
     1021 agctctgact aaacactgtg ccccaaagtg ttgaggaatt gggaaaacct agctgagtta
     1081 gtggtctgtt ttctgttaca ataaagctca taatgaaaat tagccttctt tgttcttccc
     1141 caagtctttc tttctagacg aaactacttt caactgtttt aacttcctta ctgttaactt
     1201 ccatatttct agatagtatg gtcagactgt tatttcttga cttttaaatg taagatatta
     1261 tctactgact tccttctatg taagatgagg attgagctct cttacccttc ccctgaattc
     1321 gcatctagac tgatgagacg tggtagagcc acaaacagcc ggtacaagca acgatctcca
     1381 ggaccatctg aatcatgcgc ggatgacacg aactcacgac ggcgatcaca gacattaacc
     1441 cacagtacag acactgcgac aacgtggcaa ttcgtcgcaa tacaacggaa agtgaaacgt
     1501 gatttcatgc gtcattttga acattttgta aatcttattt aataatgtgt gcggcaattc
     1561 acatttaatt tatgaatgtt ttcttaacat cgcggcaact caagaaacgg caggttcgga
     1621 tcttagctac tagagaaaga ggagaaatac tagatgcgta aaggcgaaga gctgttcact
     1681 ggtgtcgtcc ctattctggt ggaactggat ggtgatgtca acggtcataa gttttccgtg
     1741 cgtggcgagg gtgaaggtga cgcaactaat ggtaaactga cgctgaagtt catctgtact
     1801 actggtaaac tgccggttcc ttggccgact ctggtaacga cgctgactta tggtgttcag
     1861 tgctttgctc gttatccgga ccatatgaag cagcatgact tcttcaagtc cgccatgccg
     1921 gaaggctatg tgcaggaacg cacgatttcc tttaaggatg acggcacgta caaaacgcgt
     1981 gcggaagtga aatttgaagg cgataccctg gtaaaccgca ttgagctgaa aggcattgac
     2041 tttaaagagg acggcaatat cctgggccat aagctggaat acaattttaa cagccacaat
     2101 gtttacatca ccgccgataa acaaaaaaat ggcattaaag cgaattttaa aattcgccac
     2161 aacgtggagg atggcagcgt gcagctggct gatcactacc agcaaaacac tccaatcggt
     2221 gatggtcctg ttctgctgcc agacaatcac tatctgagca cgcaaagcgt tctgtctaaa
     2281 gatccgaacg agaaacgcga tcatatggtt ctgctggagt tcgtaaccgc agcgggcatc
     2341 acgcatggta tggatgaact gtacaaatga ccaggcatca aataaaacga aaggctcagt
     2401 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc
     2461 acactggctc accttcgggt gggcctttct gcgtttatag ctggaaatct gctcgtcagt
     2521 ggtgctcaca ctgacgaatc atgtacagat cataccgatg actgcctggc gactcacaac
     2581 taagcaagac agccggaacc agcgccggcg aacaccactg catatatggc atatcacaac
     2641 agtccacgtc tcaagcagtt acagagatgt tacgaaccct gaacaggttg actattaatt
     2701 gtgtctgctt gatgtggaca ccaggtggcg ctggacatca gatttggaga ggcagttgtc
     2761 tagggaaccg ggctctgtgc cagcgcagga ggcaggctgg ctctcctatt ccagggatgc
     2821 tcatccagga aggaaaggtt gcatgctgga cacactaacc ttacagcggt gggctctatg
     2881 gggtacctac gtaggtacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     2941 cccccgccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     3001 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     3061 gtctatataa gcagagctcg tttagtgaac cgtcagatag ccaccatggg taaaaagcct
     3121 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtgtccgac
     3181 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
     3241 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
     3301 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaatttagc
     3361 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
     3421 gaaaccgaac tgcccgctgt tctgcaaccg gtcgcggagg ccatggatgc gatcgctgcg
     3481 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
     3541 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
     3601 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     3661 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     3721 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     3781 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     3841 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     3901 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     3961 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     4021 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     4081 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga ataactcgag
     4141 cgacgcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     4201 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     4261 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     4321 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatgagttc
     4381 ctcatccttc caacataaat atattttggg attatatcaa cattcaatgt tacttaaagt
     4441 gaccttgtaa atattttcac aactgagcca tgtttgattt gtatacttat gtttacttta
     4501 ctgtttttcc tgaagttaat aattgccttg aatttattta tttctttaaa aatgtttcat
     4561 tactcaggac tgtagtttac attacgattc tttgtgttat acagttgatg ggtttctttt
     4621 ctttcttaat ttctttaaaa aatagagatg gggtcttact atattaccca ggctggtctt
     4681 gaagtcctgg gctcaagtga tcttcctgtc tcagcctacc aagtagctga gactataggt
     4741 gcaaaaaagc cactatacct ggctagttta caggttttaa caaatgcatt atgccacgta
     4801 tccattatta caggatcaca caagatattt tcattaccct gaaagcatcc ctgtgttcca
     4861 ccaattcatc ctgcctccat gagccgctgg caaccactga tctctatagt tttgcctttt
     4921 ctaaaatgtc atataattgg aatcatacag tctgtagcat tttcagacta gcttttaaaa
     4981 tttggcaata tgcatttaag gttcctcctt aaatgtgaag ggcatagcca atgtggcttg
     5041 atagctcatt tctttttatt ggtgaatatt tcattgtctg gatgttccac agtttgttta
     5101 tccattcacc tattcaattt gctttttttc tgtgtatcta tcactaattc aatactggac
     5161 tctccaacag agcccactta tatgaggtac ctggagtagt cagactgata gagacagaaa
     5221 ctagattggt ggttgccagg ggcagagagg aaggggaatg aggagttatt gcttcaagaa
     5281 tacaaagttt cactttttaa gatgaaaggt tttctggagg tagatggtgg tgccgagcgg
     5341 ccgcgtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa
     5401 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     5461 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     5521 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     5581 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat
     5641 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     5701 catcaaccaa accgttattc attcgtgatt gcgcctgagc gaggcgaaat acgcgatcgc
     5761 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     5821 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     5881 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     5941 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     6001 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     6061 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     6121 aatcagcatc catgttggaa tttaatcgcg gcctggagca agacgtttcc cgttgaatat
     6181 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg
     6241 atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg tggctttgtt
     6301 gaataaatcg aacttttgct gagttgaagg atcagtcatg accaaaatcc cttaacgtga
     6361 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6421 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6481 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6541 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6601 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6661 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6721 gtcgggctga acggggggtt cgtgcacaca gccca
//
