LOCUS       sequence-236793-        7576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the cROSA26 PhiC31 landing pad 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 7576)
  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 7576)
  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_033
FEATURES             Location/Qualifiers
     source          1..7576
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    209..242
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(889..1704)
                     /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(889..1704)
                     /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     1612..1631
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1696..1715
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1929..2517
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2418..2437
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2633..2649)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2633..2649)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    3053..3086
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    3704..3737
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        3759..4062
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4064..4339
                     /label=chicken beta-actin promoter
     intron          4340..5352
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     5276..5298
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     5360..5379
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             5464..6483
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             5464..6483
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             6499..6552
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6559..6579
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6595..7302
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
     primer_bind     complement(7463..7482)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7517..7536
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 gttacagaga tgttacgaac cctgaacagg ttgactatta attgtgtctg cttgatgtgg
       61 acaccaggtg gcgctggaca tcagatttgg agaggcagtt gtctagggaa ccgggctctg
      121 tgccagcgca ggaggcaggc tggctctcct attccaggga tgctcatcca ggaaggaaag
      181 gttgcatgct ggacacacta accttacaga agttcctatt ctctagaaag tataggaact
      241 tcgagtagat cgcgggggag ggagggattc ttctaggccc agggcggtcc ttaggagaca
      301 ggaggcagcc gagaactccc ataaaggtat tgcggcactc ccctccccct accgagaagg
      361 gtgcggcctt ctctccgcct cctccactgc agatccctca ggattgcagc tcgcgccgat
      421 tttcagagat cacgcacctc cctcccacaa accagcagga ccgacccccg ctcctccccc
      481 cacctccgcg agtccgcgag cggatccagc tcatctcgtc cttcaggcgg cggacgcccg
      541 ggacggagcc gcggtcgccg ccacccagaa gcctcgaccc agcagtccgc ctccgcctcc
      601 gcctccgcct ctagtgcgtg cgcaggggcg cggggccaga cactgcggcg cggggcctcg
      661 ggaaggaccg taccaacgac cgcctccctg cccctccgcc ccctctgctg acccgctcag
      721 tggtttcgct caattgggac ccgagccaat ggcaaggggc cgttccccgg cttcagcagc
      781 caatcaggtg ccgaagccac acgggcggcg ggtaacccta ctccccgcga gggttttccc
      841 agtcacgacc cgagcggccg cgtgttacaa ccaattaacc aattctgatt agaaaaactc
      901 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
      961 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     1021 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     1081 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     1141 gaatggcaaa agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     1201 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     1261 gcgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     1321 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     1381 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     1441 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     1501 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     1561 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     1621 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tggagcaaga
     1681 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     1741 ttttattgtt catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga
     1801 cacaacgtgg ctttgttgaa taaatcgaac ttttgctgag ttgaaggatc agtcatgacc
     1861 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     1921 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     1981 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2041 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     2101 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2161 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2221 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2281 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2341 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2401 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     2461 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2521 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     2581 tttcctgcgt tatcccctga ttctgtggat aaccgtgcgg ccgccaatca tggtcatagc
     2641 tgtttcctgc aacggaaagg gcctaaaatt ttaagattag tatctaattt aaaatgctac
     2701 actttggtgg tgtaaacaac gttctcaaac gacgtgagta aaaaactcag atgaccatgc
     2761 ccttaacctc cttttatcca caggaaaagc actacaaaaa cgatcttaag tagtaataac
     2821 acgcagcctt gctgcataaa atcagcccga gatgaatgcc tggactccca tttccctcct
     2881 ccacccctat tataattatt ttgtgacaca aactcaagca gggtgaagag agaaatctca
     2941 cacctgctca atcccctgca ggacaacgcc cagacaccag gtaaagcctg ggcacagaga
     3001 gtcccgccca caatcgagaa ggagcaactc gacatggagg cgatgacgcc ctgaagttcc
     3061 tattctctag aaagtatagg aacttcctga actggccgat aattgcagac gatcgaattc
     3121 gagttggcgc gcctgtcatt ctaaatctct ctttcagcct aaagcttttt ccccgtatcc
     3181 ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg
     3241 ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc
     3301 gccggaccgg agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta
     3361 attacatccc tgggggcttt gggagggggc tgtccccgtg agctcccagg cgcgcctgtc
     3421 attctaaatc tctctttcag cctaaagctt tttccccgta tccccccagg tgtctgcagg
     3481 ctcaaagagc agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc
     3541 gggctgtccc cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag
     3601 ccccgggcgg ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc
     3661 tttggggggg ggctgtcccc gtgagctcaa taaaagagcc cacataactt cgtataatgt
     3721 atgctatacg aagttataac gccatatatg gagttccgcg ttacataact tacggtaaat
     3781 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     3841 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     3901 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     3961 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     4021 acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt gagccccacg
     4081 ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt
     4141 ttttaattat tttgtgcagc gatgggggcg gggggggggg gggcgcgcgc caggcggggc
     4201 ggggcggggc gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag
     4261 cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
     4321 gcgaagcgcg cggcgggcgg gagtcgctgc gttgccttcg ccccgtgccc cgctccgcgc
     4381 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
     4441 ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc tcgtttcttt
     4501 tctgtggctg cgtgaaagcc ttaaagggct ccgggagggc cctttgtgcg ggggggagcg
     4561 gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc ccgcgctgcc
     4621 cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgcg tgtgcgcgag
     4681 gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg ctgcgagggg aacaaaggct
     4741 gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcggcg gtcgggctgt
     4801 aacccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg
     4861 ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg
     4921 gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc
     4981 ccccggagtg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa
     5041 tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg
     5101 aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag
     5161 gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag
     5221 cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc
     5281 ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc
     5341 tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa
     5401 gaattcgcca ctatggcccc aactggggta acctttgagt tctctcagtt ggggggttct
     5461 ggtaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     5521 agcgtgtccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     5581 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     5641 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     5701 ggggaattta gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     5761 caagacctgc ctgaaaccga actgcccgct gttctgcaac cggtcgcgga ggccatggat
     5821 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     5881 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     5941 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     6001 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     6061 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     6121 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     6181 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     6241 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     6301 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     6361 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     6421 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     6481 gaaggatccg gaagcggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag
     6541 aatcctggac ctggatcccc caagaagaag aggaaggtgg aggacgccgg atccgtgtcc
     6601 aaaggagagg agttaatcaa ggaaaacatg agaatgaaag ttgtcatgga gggctccgtt
     6661 aatggtcacc aattcaagtg tacaggggaa ggtgaaggta atccttacat gggtacacaa
     6721 actatgagaa ttaaagtaat tgaaggcgga ccactaccat ttgcatttga cattctggca
     6781 acgtcattca tgtacggatc acgaactttc atcaagtacc ctaaaggtat accagacttt
     6841 ttcaagcaat cttttccaga gggttttaca tgggaaaggg ttacaagata cgaagatggg
     6901 ggtgtcgtca cagttatgca agatacttca ttagaagatg gctgccttgt ctatcatgtg
     6961 caagtaagag gggtgaattt tccttctaac ggacctgtga tgcagaaaaa gaccaaaggt
     7021 tgggaaccaa atactgaaat gatgtaccca gctgatggag gtttgagagg ctacacacac
     7081 atggcgctta aagttgatgg tggaggtcat ttgtcttgta gttttgttac cacttatcgt
     7141 tctaaaaaga ctgttggcaa tatcaaaatg ccaggaatac atgctgtaga ccacagacta
     7201 gaaagactcg aagagagcga taacgaaatg ttcgttgtac agagagagca tgccgtagcc
     7261 aaatttgctg gcttaggcgg tggtatggat gaattgtata agggatccca tggcttcccg
     7321 ccggaggtgg aggagcagga tgatggcgcg ctgcccatgt cttgtgccca ggagagcggg
     7381 atggaccgtc accctgcagc ctgtgcttct gctaggatca atgtgtaact cgagtggcga
     7441 tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     7501 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     7561 caataaacgc tgagca
//
