LOCUS       sequence-251797-        6616 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the cROSA26 destination with hygyomycin resistance cassette 
            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 6616)
  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 6616)
  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_045
FEATURES             Location/Qualifiers
     source          1..6616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             149..160
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             167..880
                     /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      892..963
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      979..1006
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2134..2337
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2288..2308
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2359..2368
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2365..3393
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2365..3393
                     /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(3405..3422)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3411..3618
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(4280..5095)
                     /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(4280..5095)
                     /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     5003..5022
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5087..5106
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5320..5908
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5809..5828
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6024..6040)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6024..6040)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gaaagtgaaa cgtgatttca tgcgtcattt tgaacatttt gtaaatctta tttaataatg
       61 tgtgcggcaa ttcacattta atttatgaat gttttcttaa catcgcggca actcaagaaa
      121 cggcaggttc ggatcttagc tactagagaa agaggagaaa tactagatgc gtaaaggcga
      181 agagctgttc actggtgtcg tccctattct ggtggaactg gatggtgatg tcaacggtca
      241 taagttttcc gtgcgtggcg agggtgaagg tgacgcaact aatggtaaac tgacgctgaa
      301 gttcatctgt actactggta aactgccggt tccttggccg actctggtaa cgacgctgac
      361 ttatggtgtt cagtgctttg ctcgttatcc ggaccatatg aagcagcatg acttcttcaa
      421 gtccgccatg ccggaaggct atgtgcagga acgcacgatt tcctttaagg atgacggcac
      481 gtacaaaacg cgtgcggaag tgaaatttga aggcgatacc ctggtaaacc gcattgagct
      541 gaaaggcatt gactttaaag aggacggcaa tatcctgggc cataagctgg aatacaattt
      601 taacagccac aatgtttaca tcaccgccga taaacaaaaa aatggcatta aagcgaattt
      661 taaaattcgc cacaacgtgg aggatggcag cgtgcagctg gctgatcact accagcaaaa
      721 cactccaatc ggtgatggtc ctgttctgct gccagacaat cactatctga gcacgcaaag
      781 cgttctgtct aaagatccga acgagaaacg cgatcatatg gttctgctgg agttcgtaac
      841 cgcagcgggc atcacgcatg gtatggatga actgtacaaa tgaccaggca tcaaataaaa
      901 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
      961 ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tagctggaaa
     1021 tctgctcgtc agtggtgctc acactgacga atcatgtaca gatcataccg atgactgcct
     1081 ggcgactcac aactaagcaa gacagccgga accagcgccg gcgaacacca ctgcatatat
     1141 ggcatatcac aacagtccac gtctcaagca gttacagaga tgttacgaac cactagtgca
     1201 ctgcagatcg aattcgagtt ggcgcgcctg tcattctaaa tctctctttc agcctaaagc
     1261 tttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag
     1321 gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg
     1381 ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag
     1441 cgggggaggg acgtaattac atccctgggg gctttgggag ggggctgtcc ccgtgagctc
     1501 ccaggcgcgc ctgtcattct aaatctctct ttcagcctaa agctttttcc ccgtatcccc
     1561 ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg atcccgtgcc
     1621 accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg gggggagcgc
     1681 cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga gggacgtaat
     1741 tacatccctg ggggctttgg gggggggctg tccccgtgag ctcaataaaa gagcccacta
     1801 cagcggtggg ctctatgggg tacctacgta ggtacataac ttacggtaaa tggcccgcct
     1861 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     1921 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     1981 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     2041 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     2101 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     2161 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     2221 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     2281 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
     2341 ttagtgaacc gtcagatagc caccatgggt aaaaagcctg aactcaccgc gacgtctgtc
     2401 gagaagtttc tgatcgaaaa gttcgacagc gtgtccgacc tgatgcagct ctcggagggc
     2461 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat
     2521 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg
     2581 ctcccgattc cggaagtgct tgacattggg gaatttagcg agagcctgac ctattgcatc
     2641 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt
     2701 ctgcaaccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc
     2761 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata
     2821 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt
     2881 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc
     2941 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata
     3001 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac
     3061 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg
     3121 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt
     3181 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt
     3241 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc
     3301 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga
     3361 cgccccagca ctcgtccgag ggcaaaggaa taactcgagc gacgcctcga ctgtgccttc
     3421 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3481 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3541 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3601 tagcaggcat gctggggatg cggtgggctc tatgagtaga tcgcggggga gggagggatt
     3661 cttctaggcc cagggcggtc cttaggagac aggaggcagc cgagaactcc cataaaggta
     3721 ttgcggcact cccctccccc taccgagaag ggtgcggcct tctctccgcc tcctccactg
     3781 cagatccctc aggattgcag ctcgcgccga ttttcagaga tcacgcacct ccctcccaca
     3841 aaccagcagg accgaccccc gctcctcccc ccacctccgc gagtccgcga gcggatccag
     3901 ctcatctcgt ccttcaggcg gcggacgccc gggacggagc cgcggtcgcc gccacccaga
     3961 agcctcgacc cagcagtccg cctccgcctc cgcctccgcc tctagtgcgt gcgcaggggc
     4021 gcggggccag acactgcggc gcggggcctc gggaaggacc gtaccaacga ccgcctccct
     4081 gcccctccgc cccctctgct gacccgctca gtggtttcgc tcaattggga cccgagccaa
     4141 tggcaagggg ccgttccccg gcttcagcag ccaatcaggt gccgaagcca cacgggcggc
     4201 gggtaaccct actccccgcg agggttttcc cagtcacgac ccgagcggcc gcgtgttaca
     4261 accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat
     4321 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     4381 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     4441 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     4501 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc
     4561 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     4621 cgttattcat tcgtgattgc gcctgagcga ggcgaaatac gcgatcgctg ttaaaaggac
     4681 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     4741 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     4801 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     4861 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     4921 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     4981 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     5041 tgttggaatt taatcgcggc ctggagcaag acgtttcccg ttgaatatgg ctcataacac
     5101 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat
     5161 cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa
     5221 cttttgctga gttgaaggat cagtcatgac caaaatccct taacgtgagt tttcgttcca
     5281 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5341 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5401 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5461 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5521 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5581 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5641 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5701 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5761 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5821 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5881 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5941 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6001 taaccgtgcg gccgccaatc atggtcatag ctgtttcctg caacggaaag ggcctaaaat
     6061 tttaagatta gtatctaatt taaaatgcta cactttggtg gtgtaaacaa cgttctcaaa
     6121 cgacgtgagt aaaaaactca gatgaccatg cccttaacct ccttttatcc acaggaaaag
     6181 cactacaaaa acgatcttaa gtagtaataa cacgcagcct tgctgcataa aatcagcccg
     6241 agatgaatgc ctggactccc atttccctcc tccaccccta ttataattat tttgtgacac
     6301 aaactcaagc agggtgaaga gagaaatctc acacctgctc aatcccctgc aggacaacgc
     6361 ccagacacca ggtaaagcct gggcacagag agtcccgccc acaatcgaga aggagcaact
     6421 cgacatggag gcgatgacgc cctgaattcg catctagact gatgagacgt ggtagagcca
     6481 caaacagccg gtacaagcaa cgatctccag gaccatctga atcatgcgcg gatgacacga
     6541 actcacgacg gcgatcacag acattaaccc acagtacaga cactgcgaca acgtggcaat
     6601 tcgtcgcaat acaacg
//
