LOCUS       sequence-251798-        7077 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the Cas9 Thal5.10-2_hg18 homology destination with 
            Hygromycin resistance cassette GFP dropout destination vector as a 
            type 0 part to be used in the MTK systemh.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7077)
  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 7077)
  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_046
FEATURES             Location/Qualifiers
     source          1..7077
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      702..773
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      789..816
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        1520..1723
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1674..1694
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1745..1754
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1751..2779
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1751..2779
                     /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(2791..2808)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2797..3004
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(4017..4832)
                     /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(4017..4832)
                     /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     4740..4759
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4824..4843
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5057..5645
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5546..5565
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             7036..7047
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(7054..7077,1..690)
                     /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"
ORIGIN
        1 actggtgtcg tccctattct ggtggaactg gatggtgatg tcaacggtca taagttttcc
       61 gtgcgtggcg agggtgaagg tgacgcaact aatggtaaac tgacgctgaa gttcatctgt
      121 actactggta aactgccggt tccttggccg actctggtaa cgacgctgac ttatggtgtt
      181 cagtgctttg ctcgttatcc ggaccatatg aagcagcatg acttcttcaa gtccgccatg
      241 ccggaaggct atgtgcagga acgcacgatt tcctttaagg atgacggcac gtacaaaacg
      301 cgtgcggaag tgaaatttga aggcgatacc ctggtaaacc gcattgagct gaaaggcatt
      361 gactttaaag aggacggcaa tatcctgggc cataagctgg aatacaattt taacagccac
      421 aatgtttaca tcaccgccga taaacaaaaa aatggcatta aagcgaattt taaaattcgc
      481 cacaacgtgg aggatggcag cgtgcagctg gctgatcact accagcaaaa cactccaatc
      541 ggtgatggtc ctgttctgct gccagacaat cactatctga gcacgcaaag cgttctgtct
      601 aaagatccga acgagaaacg cgatcatatg gttctgctgg agttcgtaac cgcagcgggc
      661 atcacgcatg gtatggatga actgtacaaa tgaccaggca tcaaataaaa cgaaaggctc
      721 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga
      781 gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tagctggaaa tctgctcgtc
      841 agtggtgctc acactgacga atcatgtaca gatcataccg atgactgcct ggcgactcac
      901 aactaagcaa gacagccgga accagcgccg gcgaacacca ctgcatatat ggcatatcac
      961 aacagtccac gtctcaagca gttacagaga tgttacgaac cctgaacagg ttgactatta
     1021 attgtgtctg cttgatgtgg acaccaggtg gcgctggaca tcagatttgg agaggcagtt
     1081 gtctagggaa ccgggctctg tgccagcgca ggaggcaggc tggctctcct attccaggga
     1141 tgctcatcca ggaaggaaag gttgcatgct ggacacacta accttacagc ggtgggctct
     1201 atggggtacc tacgtaggta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     1261 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     1321 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     1381 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     1441 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     1501 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     1561 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     1621 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     1681 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca
     1741 gatagccacc atgggtaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat
     1801 cgaaaagttc gacagcgtgt ccgacctgat gcagctctcg gagggcgaag aatctcgtgc
     1861 tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg
     1921 tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga
     1981 agtgcttgac attggggaat ttagcgagag cctgacctat tgcatctccc gccgtgcaca
     2041 gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc aaccggtcgc
     2101 ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt
     2161 cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga
     2221 tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca
     2281 ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca
     2341 cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga
     2401 ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag
     2461 gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct
     2521 tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca
     2581 gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat
     2641 cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt
     2701 ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg
     2761 tccgagggca aaggaataac tcgagcgacg cctcgactgt gccttctagt tgccagccat
     2821 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     2881 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     2941 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     3001 gggatgcggt gggctctatg agtgtttata cagttgcctt acaaagacag tattagaata
     3061 aaaaaagcct caggcacttc tattataata atgaatgtct catgagatgg gatgatgtgc
     3121 aggttacaac attctgatag taatttctaa tgcattacag tagagcatgg actcttgaga
     3181 atcagatgaa attttcttcc atctagatta cattttctca ctgtcccaat gtgcttgata
     3241 tgaactgaca ccaccataaa tacttttatc tgaatggcag cactgtcata gttagacaga
     3301 gaaatatata gcgttgagtg aaaaataatt ttagatactt tccaagtgct ttttgctgtt
     3361 gtaacagtaa ttcctgcaaa gtacatacta aaccatgtaa aattacaatc ctgactattg
     3421 agttagtgaa ttatgtatac tttgaggaaa gcggaaaatg tgtattcaga tagtatgtaa
     3481 gatagctgcg ctttctggag gcttatagat ctacatattg cctatgtaat cactcgagga
     3541 aggaaaccaa gtgctcttgc ccttctaatg aattttgaat gcatcagtaa tcactgtatt
     3601 atgtatctcc aattaactag agaaacaggc tcacaatatt ctaaatttgt acactaagga
     3661 agtacataac actgtgttgt taattgagaa aatactcatt ccaacaaggt taacaaccct
     3721 aaaggagtta aaatttatca agtgcttgaa caatgcatag cacttattaa gtgctaaaca
     3781 atttgttgac aattaaatat tcactgtttg aattttgtaa attgcatttt cttcataaaa
     3841 cactgcataa atttgtctca tattttaaat tatgactttc ctttttttct tcaatgagtc
     3901 taaataacat tggtggtgtt agatatgatt gaactctatt attgattgaa ttcactaagc
     3961 aaattatcca gctttttccg agcggccgcg tgttacaacc aattaaccaa ttctgattag
     4021 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     4081 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     4141 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     4201 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     4261 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca
     4321 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     4381 tgagcgaggc gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     4441 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     4501 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     4561 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     4621 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     4681 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     4741 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctg
     4801 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     4861 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
     4921 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag
     4981 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     5041 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5101 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5161 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     5221 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5281 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5341 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5401 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5461 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5521 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5581 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5641 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5701 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtgcggcc gccaattaga
     5761 aatgtatgaa ttaaattgga ttaatcacaa tatatactat ataaaaaagt atgcagtaca
     5821 taaatacaca gatacaaaat ataggcatca tttttgaagt tatgttataa tatattgagg
     5881 ctattttatt agaaaacgaa ctttatgcta actgtataaa ggaaaaaaga ataattgcag
     5941 aagaaataat ttataatttg cttttccttg gttttattta aaaattaaat taattaaatt
     6001 ataagtatat tgtttccata taatttataa taaataaggc atataggcca ggcacggtgg
     6061 ctcatgcctg taatcccagc actttgggag gcagaggtgg gtgaacgcct gaggtcaggc
     6121 gttcaagacc agcctggcca acatggtgaa acctcgtctg tacaaaaaat acaaaaatca
     6181 gctgggcatg gtgtcagatg cctgtaatcc caggtacttg ggaggctgag gcaggagaat
     6241 ctcttgaacc caggaggcag aggttgcagt gagccgagat catgccattg cactcctgcc
     6301 tgggggacaa gagcgagact tcgtctaaaa aacaaaacaa aacaaaacaa aaacaaaaaa
     6361 aaacacaaag aaacaaaaaa ccatttaaag caacaaatta ctcatttctt agctatattc
     6421 tgtttaatta aaaaataggg tatcctctat gtaaagaaaa atataatgtt attgagccat
     6481 tttcttcaga agaacaagct actctaccta tgtagagtga taatgtatcc tcaggattga
     6541 agatcaaaag aggtaatata tacaaacatt ctttgaaagg tgccttataa aacaaaaaat
     6601 tgttttatca taaaaattta aggtagtgca gagagctaac atgaaaaatg taaacattgg
     6661 caataagaac aaagatatac tatgtttgaa aaatggttga agtccctttc ccctgaattc
     6721 gcatctagac tgatgagacg tggtagagcc acaaacagcc ggtacaagca acgatctcca
     6781 ggaccatctg aatcatgcgc ggatgacacg aactcacgac ggcgatcaca gacattaacc
     6841 cacagtacag acactgcgac aacgtggcaa ttcgtcgcaa tacaacggaa agtgaaacgt
     6901 gatttcatgc gtcattttga acattttgta aatcttattt aataatgtgt gcggcaattc
     6961 acatttaatt tatgaatgtt ttcttaacat cgcggcaact caagaaacgg caggttcgga
     7021 tcttagctac tagagaaaga ggagaaatac tagatgcgta aaggcgaaga gctgttc
//
