LOCUS       sequence-251793-        7592 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Encodes the cROSA26 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 7592)
  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 7592)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_036
FEATURES             Location/Qualifiers
     source          1..7592
                     /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..5356
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     5280..5302
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     5364..5383
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    5422..5469
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             5480..6499
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             5480..6499
                     /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             6515..6568
                     /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             6575..6595
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6611..7318
                     /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(7479..7498)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7533..7552
                     /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 gggggcgcgc caggcggggc
     4201 ggggcggggc gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag
     4261 cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
     4321 gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
     4381 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg
     4441 gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc
     4501 ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag
     4561 cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct
     4621 gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg
     4681 cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa
     4741 aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg
     4801 gctgcaaccc cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc
     4861 ggggctccgt acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt
     4921 gggggtgccg ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg
     4981 cggcccccgg agtgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg
     5041 gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc
     5101 tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag
     5161 gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct
     5221 ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg
     5281 gttcggcttc tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt
     5341 cttctttttc ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg
     5401 caaagaattc gccactatgg tggtttgtct ggtcaaccac cgcggtctca gtggtgtacg
     5461 gtacaaacca ggttctggta aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     5521 gatcgaaaag ttcgacagcg tgtccgacct gatgcagctc tcggagggcg aagaatctcg
     5581 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     5641 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
     5701 ggaagtgctt gacattgggg aatttagcga gagcctgacc tattgcatct cccgccgtgc
     5761 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcaaccggt
     5821 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     5881 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
     5941 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     6001 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     6061 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     6121 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     6181 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     6241 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     6301 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     6361 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     6421 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     6481 tcgtccgagg gcaaaggaag gatccggaag cggagagggc agaggaagtc tgctaacatg
     6541 cggtgacgtc gaggagaatc ctggacctgg atcccccaag aagaagagga aggtggagga
     6601 cgccggatcc gtgtccaaag gagaggagtt aatcaaggaa aacatgagaa tgaaagttgt
     6661 catggagggc tccgttaatg gtcaccaatt caagtgtaca ggggaaggtg aaggtaatcc
     6721 ttacatgggt acacaaacta tgagaattaa agtaattgaa ggcggaccac taccatttgc
     6781 atttgacatt ctggcaacgt cattcatgta cggatcacga actttcatca agtaccctaa
     6841 aggtatacca gactttttca agcaatcttt tccagagggt tttacatggg aaagggttac
     6901 aagatacgaa gatgggggtg tcgtcacagt tatgcaagat acttcattag aagatggctg
     6961 ccttgtctat catgtgcaag taagaggggt gaattttcct tctaacggac ctgtgatgca
     7021 gaaaaagacc aaaggttggg aaccaaatac tgaaatgatg tacccagctg atggaggttt
     7081 gagaggctac acacacatgg cgcttaaagt tgatggtgga ggtcatttgt cttgtagttt
     7141 tgttaccact tatcgttcta aaaagactgt tggcaatatc aaaatgccag gaatacatgc
     7201 tgtagaccac agactagaaa gactcgaaga gagcgataac gaaatgttcg ttgtacagag
     7261 agagcatgcc gtagccaaat ttgctggctt aggcggtggt atggatgaat tgtataaggg
     7321 atcccatggc ttcccgccgg aggtggagga gcaggatgat ggcgcgctgc ccatgtcttg
     7381 tgcccaggag agcgggatgg accgtcaccc tgcagcctgt gcttctgcta ggatcaatgt
     7441 gtaactcgag tggcgatcca gacatgataa gatacattga tgagtttgga caaaccacaa
     7501 ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg
     7561 taaccattat aagctgcaat aaacgctgag ca
//
