LOCUS       sequence-251672-        6721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the hROSA26 PhiC31 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 6721)
  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 6721)
  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_011
FEATURES             Location/Qualifiers
     source          1..6721
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(48..863)
                     /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(48..863)
                     /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     771..790
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     855..874
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1088..1676
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1577..1596
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2246..2279
                     /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    2897..2930
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2947..3457
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3376..3394
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3510..4529
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3510..4529
                     /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             4545..4598
                     /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             4605..4625
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4641..5348
                     /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(5367..5384)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5373..5597
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    6238..6271
                     /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     join(6713..6721,1..8)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cggccagtcc gagcggccgc gtgttacaac caattaacca attctgatta gaaaaactca
       61 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
      121 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
      181 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
      241 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
      301 aatggcaaaa gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
      361 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgagg
      421 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
      481 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
      541 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
      601 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
      661 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
      721 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
      781 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct ggagcaagac
      841 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
      901 tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac
      961 acaacgtggc tttgttgaat aaatcgaact tttgctgagt tgaaggatca gtcatgacca
     1021 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     1081 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     1141 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     1201 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     1261 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     1321 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     1381 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     1441 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     1501 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     1561 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     1621 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     1681 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     1741 ttcctgcgtt atcccctgat tctgtggata accgtgcggc cgccaatcat ggtcatagct
     1801 gtttccgggg agagagggtg agggagggaa gcactggttt ctcaagcaaa agctaaaatt
     1861 tttctattaa gatttaacct gatgctacac tttggtggtg cagcaagggt ctaaaatggt
     1921 ataaaactca ggtgatcatg ctttatgtct gtctctagaa aaatgctcca aaaatgataa
     1981 gtagtgataa tccgcagtct cgttgcataa aatcagcgcc aggtgaatga ctaagctcca
     2041 tttccctacc ccacccttat tacaataacc tcgacaccaa ctctagtccg tgcgaagata
     2101 aactaatcgg agtcgcccct caaatcttac agctgctcac tcccctgcag ggcaacgccc
     2161 agggaccaag ttagcccctt aagcctaggc aaaagaatcc cgcccataat cgagaagcga
     2221 ctcgacatgg aggcgatgac gccctgaagt tcctattctc tagaaagtat aggaacttcc
     2281 tgaactggcc gataattgca gacgatcgaa ttcgagttgg cgcgcctgtc attctaaatc
     2341 tctctttcag cctaaagctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc
     2401 agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc
     2461 cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg
     2521 ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttgggaggg
     2581 ggctgtcccc gtgagctccc aggcgcgcct gtcattctaa atctctcttt cagcctaaag
     2641 ctttttcccc gtatcccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga
     2701 ggaaagcgat cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg
     2761 gggatgcggg gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta
     2821 gcgggggagg gacgtaatta catccctggg ggctttgggg gggggctgtc cccgtgagct
     2881 caataaaaga gcccacataa cttcgtataa tgtatgctat acgaagttat aacgtcgaat
     2941 tcccacgggg ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg
     3001 ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggtctt gcacattctt
     3061 cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac
     3121 gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg
     3181 acaaacggaa gccgcacgtg tcactagtac cctcgcagac ggacagcgcc agggagcaat
     3241 ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga
     3301 gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct
     3361 gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg
     3421 ctccctcgtt gaccgaatca ccgacctctc tccccagtat ggccccaact ggggtaacct
     3481 ttgagttctc tcagttgggg ggttctggta aaaagcctga actcaccgcg acgtctgtcg
     3541 agaagtttct gatcgaaaag ttcgacagcg tgtccgacct gatgcagctc tcggagggcg
     3601 aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata
     3661 gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc
     3721 tcccgattcc ggaagtgctt gacattgggg aatttagcga gagcctgacc tattgcatct
     3781 cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc
     3841 tgcaaccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg
     3901 ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat
     3961 gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg
     4021 cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc
     4081 ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa
     4141 cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca
     4201 tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga
     4261 ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg
     4321 accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc
     4381 gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca
     4441 gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac
     4501 gccccagcac tcgtccgagg gcaaaggaag gatccggaag cggagagggc agaggaagtc
     4561 tgctaacatg cggtgacgtc gaggagaatc ctggacctgg atcccccaag aagaagagga
     4621 aggtggagga cgccggatcc gtgtccaaag gagaggagtt aatcaaggaa aacatgagaa
     4681 tgaaagttgt catggagggc tccgttaatg gtcaccaatt caagtgtaca ggggaaggtg
     4741 aaggtaatcc ttacatgggt acacaaacta tgagaattaa agtaattgaa ggcggaccac
     4801 taccatttgc atttgacatt ctggcaacgt cattcatgta cggatcacga actttcatca
     4861 agtaccctaa aggtatacca gactttttca agcaatcttt tccagagggt tttacatggg
     4921 aaagggttac aagatacgaa gatgggggtg tcgtcacagt tatgcaagat acttcattag
     4981 aagatggctg ccttgtctat catgtgcaag taagaggggt gaattttcct tctaacggac
     5041 ctgtgatgca gaaaaagacc aaaggttggg aaccaaatac tgaaatgatg tacccagctg
     5101 atggaggttt gagaggctac acacacatgg cgcttaaagt tgatggtgga ggtcatttgt
     5161 cttgtagttt tgttaccact tatcgttcta aaaagactgt tggcaatatc aaaatgccag
     5221 gaatacatgc tgtagaccac agactagaaa gactcgaaga gagcgataac gaaatgttcg
     5281 ttgtacagag agagcatgcc gtagccaaat ttgctggctt aggcggtggt atggatgaat
     5341 tgtataagta actcgagtgg ccgacgcctc gactgtgcct tctagttgcc agccatctgt
     5401 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     5461 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     5521 tggggtggtg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     5581 tgcggtgggc tctatgggct gagcagttac agagatgtta cgaaccacta gtgcactgca
     5641 gatcgaattc gagttggcgc gcctgtcatt ctaaatctct ctttcagcct aaagcttttt
     5701 ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag
     5761 cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg
     5821 cggggggagc gccggaccgg agcggagccc cgggcggctc gctgctgccc cctagcgggg
     5881 gagggacgta attacatccc tgggggcttt gggagggggc tgtccccgtg agctcccagg
     5941 cgcgcctgtc attctaaatc tctctttcag cctaaagctt tttccccgta tccccccagg
     6001 tgtctgcagg ctcaaagagc agcgagaagc gttcagagga aagcgatccc gtgccacctt
     6061 ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg atgcgggggg agcgccggac
     6121 cggagcggag ccccgggcgg ctcgctgctg ccccctagcg ggggagggac gtaattacat
     6181 ccctgggggc tttggggggg ggctgtcccc gtgagctcaa taaaagagcc cactacagaa
     6241 gttcctattc tctagaaagt ataggaactt cgagtggagg cagcagagaa ctcccataaa
     6301 ggtattgcgg cactcccctc cccctgccca gaagggtgcg gccttctctc cacctcctcc
     6361 accgcagctc cctcaggatt gcagctcgcg ccggtttttg gagaacaagc gcctcccacc
     6421 cacaaaccag ccggaccgac ccccgctcct cccccacccc cacgagtgcc tgtagcaggt
     6481 cgggcttgtc tcgcccttca ggcggtggga acccagggcg gagcctcggc cgccgccatc
     6541 cagaagtctc ggccggcagc ccgacccctc ctccagcgcg cgcttcatgc cacgttgcgc
     6601 aggggcgcgg ggacagacac tgcggcgctc ggcgtcgggg aggaccgtac caacgcccgc
     6661 ctcccctcca cccccgcgcc ccgcgcagtg gtttcgctca tgtgagactc gagtaaaacg
     6721 a
//
