LOCUS       sequence-251720-        6733 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the hROSA26 BxBI recognition site with Hygromycin resistance
            and mRuby2 landing pad with GFP dropout destination vector 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 6733)
  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 6733)
  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_012
FEATURES             Location/Qualifiers
     source          1..6733
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    468..501
                     /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    1119..1152
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1169..1679
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1598..1616
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     protein_bind    1686..1733
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             1744..2763
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             1744..2763
                     /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             2779..2832
                     /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             2839..2859
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2875..3582
                     /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(3601..3618)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3607..3831
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    4472..4505
                     /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     4947..4963
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(5003..5818)
                     /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(5003..5818)
                     /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     5726..5745
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5810..5829
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6043..6631
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6532..6551
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gccgccaatc atggtcatag ctgtttccgg ggagagaggg tgagggaggg aagcactggt
       61 ttctcaagca aaagctaaaa tttttctatt aagatttaac ctgatgctac actttggtgg
      121 tgcagcaagg gtctaaaatg gtataaaact caggtgatca tgctttatgt ctgtctctag
      181 aaaaatgctc caaaaatgat aagtagtgat aatccgcagt ctcgttgcat aaaatcagcg
      241 ccaggtgaat gactaagctc catttcccta ccccaccctt attacaataa cctcgacacc
      301 aactctagtc cgtgcgaaga taaactaatc ggagtcgccc ctcaaatctt acagctgctc
      361 actcccctgc agggcaacgc ccagggacca agttagcccc ttaagcctag gcaaaagaat
      421 cccgcccata atcgagaagc gactcgacat ggaggcgatg acgccctgaa gttcctattc
      481 tctagaaagt ataggaactt cctgaactgg ccgataattg cagacgatcg aattcgagtt
      541 ggcgcgcctg tcattctaaa tctctctttc agcctaaagc tttttccccg tatcccccca
      601 ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc
      661 ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg
      721 accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac
      781 atccctgggg gctttgggag ggggctgtcc ccgtgagctc ccaggcgcgc ctgtcattct
      841 aaatctctct ttcagcctaa agctttttcc ccgtatcccc ccaggtgtct gcaggctcaa
      901 agagcagcga gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct
      961 gtccccgcac gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg
     1021 ggcggctcgc tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg
     1081 gggggggctg tccccgtgag ctcaataaaa gagcccacat aacttcgtat aatgtatgct
     1141 atacgaagtt ataacgtcga attcccacgg ggttggggtt gcgccttttc caaggcagcc
     1201 ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg
     1261 accctgggtc ttgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta
     1321 cccttgtggg ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc
     1381 ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg tgtcactagt accctcgcag
     1441 acggacagcg ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg
     1501 gctgctcagc agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt
     1561 gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc
     1621 cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccagt
     1681 atggtggttt gtctggtcaa ccaccgcggt ctcagtggtg tacggtacaa accaggttct
     1741 ggtaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     1801 agcgtgtccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     1861 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     1921 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     1981 ggggaattta gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     2041 caagacctgc ctgaaaccga actgcccgct gttctgcaac cggtcgcgga ggccatggat
     2101 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     2161 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     2221 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     2281 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     2341 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     2401 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     2461 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     2521 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     2581 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     2641 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     2701 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     2761 gaaggatccg gaagcggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag
     2821 aatcctggac ctggatcccc caagaagaag aggaaggtgg aggacgccgg atccgtgtcc
     2881 aaaggagagg agttaatcaa ggaaaacatg agaatgaaag ttgtcatgga gggctccgtt
     2941 aatggtcacc aattcaagtg tacaggggaa ggtgaaggta atccttacat gggtacacaa
     3001 actatgagaa ttaaagtaat tgaaggcgga ccactaccat ttgcatttga cattctggca
     3061 acgtcattca tgtacggatc acgaactttc atcaagtacc ctaaaggtat accagacttt
     3121 ttcaagcaat cttttccaga gggttttaca tgggaaaggg ttacaagata cgaagatggg
     3181 ggtgtcgtca cagttatgca agatacttca ttagaagatg gctgccttgt ctatcatgtg
     3241 caagtaagag gggtgaattt tccttctaac ggacctgtga tgcagaaaaa gaccaaaggt
     3301 tgggaaccaa atactgaaat gatgtaccca gctgatggag gtttgagagg ctacacacac
     3361 atggcgctta aagttgatgg tggaggtcat ttgtcttgta gttttgttac cacttatcgt
     3421 tctaaaaaga ctgttggcaa tatcaaaatg ccaggaatac atgctgtaga ccacagacta
     3481 gaaagactcg aagagagcga taacgaaatg ttcgttgtac agagagagca tgccgtagcc
     3541 aaatttgctg gcttaggcgg tggtatggat gaattgtata agtaactcga gtggccgacg
     3601 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     3661 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     3721 attgtctgag taggtgtcat tctattctgg ggggtggggt ggtgcaggac agcaaggggg
     3781 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg ggctgagcag
     3841 ttacagagat gttacgaacc actagtgcac tgcagatcga attcgagttg gcgcgcctgt
     3901 cattctaaat ctctctttca gcctaaagct ttttccccgt atccccccag gtgtctgcag
     3961 gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc
     4021 cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga ccggagcgga
     4081 gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca tccctggggg
     4141 ctttgggagg gggctgtccc cgtgagctcc caggcgcgcc tgtcattcta aatctctctt
     4201 tcagcctaaa gctttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag
     4261 aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg
     4321 ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct
     4381 gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt
     4441 ccccgtgagc tcaataaaag agcccactac agaagttcct attctctaga aagtatagga
     4501 acttcgagtg gaggcagcag agaactccca taaaggtatt gcggcactcc cctccccctg
     4561 cccagaaggg tgcggccttc tctccacctc ctccaccgca gctccctcag gattgcagct
     4621 cgcgccggtt tttggagaac aagcgcctcc cacccacaaa ccagccggac cgacccccgc
     4681 tcctccccca cccccacgag tgcctgtagc aggtcgggct tgtctcgccc ttcaggcggt
     4741 gggaacccag ggcggagcct cggccgccgc catccagaag tctcggccgg cagcccgacc
     4801 cctcctccag cgcgcgcttc atgccacgtt gcgcaggggc gcggggacag acactgcggc
     4861 gctcggcgtc ggggaggacc gtaccaacgc ccgcctcccc tccacccccg cgccccgcgc
     4921 agtggtttcg ctcatgtgag actcgagtaa aacgacggcc agtccgagcg gccgcgtgtt
     4981 acaaccaatt aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt
     5041 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     5101 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     5161 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     5221 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctta tgcatttctt
     5281 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     5341 aaccgttatt cattcgtgat tgcgcctgag cgaggcgaaa tacgcgatcg ctgttaaaag
     5401 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     5461 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     5521 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     5581 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     5641 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     5701 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     5761 ccatgttgga atttaatcgc ggcctggagc aagacgtttc ccgttgaata tggctcataa
     5821 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt
     5881 tatcttgtgc aatgtaacat cagagatttt gagacacaac gtggctttgt tgaataaatc
     5941 gaacttttgc tgagttgaag gatcagtcat gaccaaaatc ccttaacgtg agttttcgtt
     6001 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6061 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6121 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6181 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6241 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6301 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6361 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6421 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6481 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6541 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6601 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6661 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6721 ggataaccgt gcg
//
