LOCUS       sequence-251792-        7889 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the hROSA26 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 7889)
  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 7889)
  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_034
FEATURES             Location/Qualifiers
     source          1..7889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     723..742
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     807..826
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1040..1628
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1529..1548
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2198..2231
                     /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    2849..2882
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        2904..3207
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3209..3484
                     /label=chicken beta-actin promoter
     intron          3485..4501
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4425..4447
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4509..4528
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             4613..5632
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4613..5632
                     /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             5648..5701
                     /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             5708..5728
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5744..6451
                     /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(6612..6631)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6666..6685
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    7358..7391
                     /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)"
     primer_bind     7833..7849
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(join(7889,1..815))
                     /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(join(7889,1..815))
                     /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"
ORIGIN
        1 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
       61 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
      121 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
      181 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
      241 gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag
      301 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
      361 gcctgagcga ggcgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
      421 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
      481 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
      541 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
      601 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
      661 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
      721 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
      781 ctggagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
      841 taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag
      901 agattttgag acacaacgtg gctttgttga ataaatcgaa cttttgctga gttgaaggat
      961 cagtcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1021 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1081 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1141 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1201 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1261 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1321 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1381 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1441 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1501 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1561 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1621 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1681 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtgcg gccgccaatc
     1741 atggtcatag ctgtttccgg ggagagaggg tgagggaggg aagcactggt ttctcaagca
     1801 aaagctaaaa tttttctatt aagatttaac ctgatgctac actttggtgg tgcagcaagg
     1861 gtctaaaatg gtataaaact caggtgatca tgctttatgt ctgtctctag aaaaatgctc
     1921 caaaaatgat aagtagtgat aatccgcagt ctcgttgcat aaaatcagcg ccaggtgaat
     1981 gactaagctc catttcccta ccccaccctt attacaataa cctcgacacc aactctagtc
     2041 cgtgcgaaga taaactaatc ggagtcgccc ctcaaatctt acagctgctc actcccctgc
     2101 agggcaacgc ccagggacca agttagcccc ttaagcctag gcaaaagaat cccgcccata
     2161 atcgagaagc gactcgacat ggaggcgatg acgccctgaa gttcctattc tctagaaagt
     2221 ataggaactt cctgaactgg ccgataattg cagacgatcg aattcgagtt ggcgcgcctg
     2281 tcattctaaa tctctctttc agcctaaagc tttttccccg tatcccccca ggtgtctgca
     2341 ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc
     2401 ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg
     2461 agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg
     2521 gctttgggag ggggctgtcc ccgtgagctc ccaggcgcgc ctgtcattct aaatctctct
     2581 ttcagcctaa agctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga
     2641 gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac
     2701 gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc
     2761 tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg
     2821 tccccgtgag ctcaataaaa gagcccacat aacttcgtat aatgtatgct atacgaagtt
     2881 ataacgccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     2941 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     3001 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     3061 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
     3121 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
     3181 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
     3241 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
     3301 gcagcgatgg gggcgggggg gggggggggg cgcgccaggc ggggcggggc ggggcgaggg
     3361 gcggggcggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
     3421 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
     3481 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
     3541 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
     3601 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
     3661 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
     3721 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
     3781 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
     3841 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
     3901 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     3961 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     4021 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
     4081 ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagtgc
     4141 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     4201 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     4261 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     4321 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     4381 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     4441 tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca
     4501 gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattcgccac
     4561 tatggcccca actggggtaa cctttgagtt ctctcagttg gggggttctg gtaaaaagcc
     4621 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtgtccga
     4681 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     4741 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     4801 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaatttag
     4861 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     4921 tgaaaccgaa ctgcccgctg ttctgcaacc ggtcgcggag gccatggatg cgatcgctgc
     4981 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     5041 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     5101 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     5161 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     5221 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     5281 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     5341 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     5401 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     5461 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     5521 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     5581 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aaggatccgg
     5641 aagcggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggacc
     5701 tggatccccc aagaagaaga ggaaggtgga ggacgccgga tccgtgtcca aaggagagga
     5761 gttaatcaag gaaaacatga gaatgaaagt tgtcatggag ggctccgtta atggtcacca
     5821 attcaagtgt acaggggaag gtgaaggtaa tccttacatg ggtacacaaa ctatgagaat
     5881 taaagtaatt gaaggcggac cactaccatt tgcatttgac attctggcaa cgtcattcat
     5941 gtacggatca cgaactttca tcaagtaccc taaaggtata ccagactttt tcaagcaatc
     6001 ttttccagag ggttttacat gggaaagggt tacaagatac gaagatgggg gtgtcgtcac
     6061 agttatgcaa gatacttcat tagaagatgg ctgccttgtc tatcatgtgc aagtaagagg
     6121 ggtgaatttt ccttctaacg gacctgtgat gcagaaaaag accaaaggtt gggaaccaaa
     6181 tactgaaatg atgtacccag ctgatggagg tttgagaggc tacacacaca tggcgcttaa
     6241 agttgatggt ggaggtcatt tgtcttgtag ttttgttacc acttatcgtt ctaaaaagac
     6301 tgttggcaat atcaaaatgc caggaataca tgctgtagac cacagactag aaagactcga
     6361 agagagcgat aacgaaatgt tcgttgtaca gagagagcat gccgtagcca aatttgctgg
     6421 cttaggcggt ggtatggatg aattgtataa gggatcccat ggcttcccgc cggaggtgga
     6481 ggagcaggat gatggcgcgc tgcccatgtc ttgtgcccag gagagcggga tggaccgtca
     6541 ccctgcagcc tgtgcttctg ctaggatcaa tgtgtaactc gagtggcgat ccagacatga
     6601 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
     6661 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacgct
     6721 gagcagttac agagatgtta cgaaccacta gtgcactgca gatcgaattc gagttggcgc
     6781 gcctgtcatt ctaaatctct ctttcagcct aaagcttttt ccccgtatcc ccccaggtgt
     6841 ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc
     6901 cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg
     6961 agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc
     7021 tgggggcttt gggagggggc tgtccccgtg agctcccagg cgcgcctgtc attctaaatc
     7081 tctctttcag cctaaagctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc
     7141 agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc
     7201 cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg
     7261 ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg
     7321 ggctgtcccc gtgagctcaa taaaagagcc cactacagaa gttcctattc tctagaaagt
     7381 ataggaactt cgagtggagg cagcagagaa ctcccataaa ggtattgcgg cactcccctc
     7441 cccctgccca gaagggtgcg gccttctctc cacctcctcc accgcagctc cctcaggatt
     7501 gcagctcgcg ccggtttttg gagaacaagc gcctcccacc cacaaaccag ccggaccgac
     7561 ccccgctcct cccccacccc cacgagtgcc tgtagcaggt cgggcttgtc tcgcccttca
     7621 ggcggtggga acccagggcg gagcctcggc cgccgccatc cagaagtctc ggccggcagc
     7681 ccgacccctc ctccagcgcg cgcttcatgc cacgttgcgc aggggcgcgg ggacagacac
     7741 tgcggcgctc ggcgtcgggg aggaccgtac caacgcccgc ctcccctcca cccccgcgcc
     7801 ccgcgcagtg gtttcgctca tgtgagactc gagtaaaacg acggccagtc cgagcggccg
     7861 cgtgttacaa ccaattaacc aattctgat
//
