LOCUS       sequence-251795-        7901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the hROSA26 BxBI 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 7901)
  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 7901)
  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_037
FEATURES             Location/Qualifiers
     source          1..7901
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    633..666
                     /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     1108..1124
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(1164..1979)
                     /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(1164..1979)
                     /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     1887..1906
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1971..1990
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2204..2792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2693..2712
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3362..3395
                     /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    4013..4046
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     enhancer        4068..4371
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4373..4648
                     /label=chicken beta-actin promoter
     intron          4649..5665
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     5589..5611
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     5673..5692
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    5731..5778
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             5789..6808
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             5789..6808
                     /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             6824..6877
                     /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             6884..6904
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6920..7627
                     /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(7788..7807)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7842..7861
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 gttacagaga tgttacgaac cactagtgca ctgcagatcg aattcgagtt ggcgcgcctg
       61 tcattctaaa tctctctttc agcctaaagc tttttccccg tatcccccca ggtgtctgca
      121 ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc
      181 ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg accggagcgg
      241 agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac atccctgggg
      301 gctttgggag ggggctgtcc ccgtgagctc ccaggcgcgc ctgtcattct aaatctctct
      361 ttcagcctaa agctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga
      421 gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac
      481 gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc
      541 tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg
      601 tccccgtgag ctcaataaaa gagcccacta cagaagttcc tattctctag aaagtatagg
      661 aacttcgagt ggaggcagca gagaactccc ataaaggtat tgcggcactc ccctccccct
      721 gcccagaagg gtgcggcctt ctctccacct cctccaccgc agctccctca ggattgcagc
      781 tcgcgccggt ttttggagaa caagcgcctc ccacccacaa accagccgga ccgacccccg
      841 ctcctccccc acccccacga gtgcctgtag caggtcgggc ttgtctcgcc cttcaggcgg
      901 tgggaaccca gggcggagcc tcggccgccg ccatccagaa gtctcggccg gcagcccgac
      961 ccctcctcca gcgcgcgctt catgccacgt tgcgcagggg cgcggggaca gacactgcgg
     1021 cgctcggcgt cggggaggac cgtaccaacg cccgcctccc ctccaccccc gcgccccgcg
     1081 cagtggtttc gctcatgtga gactcgagta aaacgacggc cagtccgagc ggccgcgtgt
     1141 tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg aaactgcaat
     1201 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     1261 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     1321 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     1381 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct
     1441 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     1501 aaaccgttat tcattcgtga ttgcgcctga gcgaggcgaa atacgcgatc gctgttaaaa
     1561 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     1621 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     1681 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     1741 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     1801 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     1861 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     1921 tccatgttgg aatttaatcg cggcctggag caagacgttt cccgttgaat atggctcata
     1981 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt
     2041 ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttg ttgaataaat
     2101 cgaacttttg ctgagttgaa ggatcagtca tgaccaaaat cccttaacgt gagttttcgt
     2161 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2221 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2281 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2341 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     2401 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     2461 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     2521 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     2581 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2641 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2701 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2761 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2821 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     2881 tggataaccg tgcggccgcc aatcatggtc atagctgttt ccggggagag agggtgaggg
     2941 agggaagcac tggtttctca agcaaaagct aaaatttttc tattaagatt taacctgatg
     3001 ctacactttg gtggtgcagc aagggtctaa aatggtataa aactcaggtg atcatgcttt
     3061 atgtctgtct ctagaaaaat gctccaaaaa tgataagtag tgataatccg cagtctcgtt
     3121 gcataaaatc agcgccaggt gaatgactaa gctccatttc cctaccccac ccttattaca
     3181 ataacctcga caccaactct agtccgtgcg aagataaact aatcggagtc gcccctcaaa
     3241 tcttacagct gctcactccc ctgcagggca acgcccaggg accaagttag ccccttaagc
     3301 ctaggcaaaa gaatcccgcc cataatcgag aagcgactcg acatggaggc gatgacgccc
     3361 tgaagttcct attctctaga aagtatagga acttcctgaa ctggccgata attgcagacg
     3421 atcgaattcg agttggcgcg cctgtcattc taaatctctc tttcagccta aagctttttc
     3481 cccgtatccc cccaggtgtc tgcaggctca aagagcagcg agaagcgttc agaggaaagc
     3541 gatcccgtgc caccttcccc gtgcccgggc tgtccccgca cgctgccggc tcggggatgc
     3601 ggggggagcg ccggaccgga gcggagcccc gggcggctcg ctgctgcccc ctagcggggg
     3661 agggacgtaa ttacatccct gggggctttg ggagggggct gtccccgtga gctcccaggc
     3721 gcgcctgtca ttctaaatct ctctttcagc ctaaagcttt ttccccgtat ccccccaggt
     3781 gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc
     3841 cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc
     3901 ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc
     3961 cctgggggct ttgggggggg gctgtccccg tgagctcaat aaaagagccc acataacttc
     4021 gtataatgta tgctatacga agttataacg ccatatatgg agttccgcgt tacataactt
     4081 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     4141 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     4201 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     4261 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     4321 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg
     4381 agccccacgt tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat
     4441 ttatttattt tttaattatt ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcc
     4501 aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcagc
     4561 caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc
     4621 ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc
     4681 ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
     4741 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg
     4801 gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg
     4861 cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg
     4921 ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg
     4981 cagtgtgcgc gaggggagcg cggccggggg cggtgccccg cggtgcgggg ggggctgcga
     5041 ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc
     5101 gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga gttgctgagc acggcccggc
     5161 ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg
     5221 gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga
     5281 ggggcgcggc ggcccccgga gtgccggcgg ctgtcgaggc gcggcgagcc gcagccattg
     5341 ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga
     5401 gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg
     5461 cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct
     5521 tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg
     5581 cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt
     5641 tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca
     5701 tcattttggc aaagaattcg ccactatggt ggtttgtctg gtcaaccacc gcggtctcag
     5761 tggtgtacgg tacaaaccag gttctggtaa aaagcctgaa ctcaccgcga cgtctgtcga
     5821 gaagtttctg atcgaaaagt tcgacagcgt gtccgacctg atgcagctct cggagggcga
     5881 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     5941 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     6001 cccgattccg gaagtgcttg acattgggga atttagcgag agcctgacct attgcatctc
     6061 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     6121 gcaaccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     6181 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     6241 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     6301 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     6361 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     6421 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     6481 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     6541 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     6601 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     6661 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     6721 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     6781 ccccagcact cgtccgaggg caaaggaagg atccggaagc ggagagggca gaggaagtct
     6841 gctaacatgc ggtgacgtcg aggagaatcc tggacctgga tcccccaaga agaagaggaa
     6901 ggtggaggac gccggatccg tgtccaaagg agaggagtta atcaaggaaa acatgagaat
     6961 gaaagttgtc atggagggct ccgttaatgg tcaccaattc aagtgtacag gggaaggtga
     7021 aggtaatcct tacatgggta cacaaactat gagaattaaa gtaattgaag gcggaccact
     7081 accatttgca tttgacattc tggcaacgtc attcatgtac ggatcacgaa ctttcatcaa
     7141 gtaccctaaa ggtataccag actttttcaa gcaatctttt ccagagggtt ttacatggga
     7201 aagggttaca agatacgaag atgggggtgt cgtcacagtt atgcaagata cttcattaga
     7261 agatggctgc cttgtctatc atgtgcaagt aagaggggtg aattttcctt ctaacggacc
     7321 tgtgatgcag aaaaagacca aaggttggga accaaatact gaaatgatgt acccagctga
     7381 tggaggtttg agaggctaca cacacatggc gcttaaagtt gatggtggag gtcatttgtc
     7441 ttgtagtttt gttaccactt atcgttctaa aaagactgtt ggcaatatca aaatgccagg
     7501 aatacatgct gtagaccaca gactagaaag actcgaagag agcgataacg aaatgttcgt
     7561 tgtacagaga gagcatgccg tagccaaatt tgctggctta ggcggtggta tggatgaatt
     7621 gtataaggga tcccatggct tcccgccgga ggtggaggag caggatgatg gcgcgctgcc
     7681 catgtcttgt gcccaggaga gcgggatgga ccgtcaccct gcagcctgtg cttctgctag
     7741 gatcaatgtg taactcgagt ggcgatccag acatgataag atacattgat gagtttggac
     7801 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     7861 ctttatttgt aaccattata agctgcaata aacgctgagc a
//
