LOCUS       sequence-250083-        5279 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the phiC31 attB GFP dropout destination vector constitutive 
            NLS::tagBFP 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 5279)
  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 5279)
  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_016
FEATURES             Location/Qualifiers
     source          1..5279
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             322..333
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             340..1053
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1065..1136
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1152..1179
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    1976..2009
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             2014..2712
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             2713..2733
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2764..2817
                     /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             2824..3222
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     complement(3234..3251)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3240..3447
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(3502..4317)
                     /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(3502..4317)
                     /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     4225..4244
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4309..4328
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4542..5130
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5031..5050
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5242..5275
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 gaattcgcat ctagactgat gagacgtggt agagccacaa acagccggta caagcaacga
       61 tctccaggac catctgaatc atgcgcggat gacacgaact cacgacggcg atcacagaca
      121 ttaacccaca gtacagacac tgcgacaacg tggcaattcg tcgcaataca acggaaagtg
      181 aaacgtgatt tcatgcgtca ttttgaacat tttgtaaatc ttatttaata atgtgtgcgg
      241 caattcacat ttaatttatg aatgttttct taacatcgcg gcaactcaag aaacggcagg
      301 ttcggatctt agctactaga gaaagaggag aaatactaga tgcgtaaagg cgaagagctg
      361 ttcactggtg tcgtccctat tctggtggaa ctggatggtg atgtcaacgg tcataagttt
      421 tccgtgcgtg gcgagggtga aggtgacgca actaatggta aactgacgct gaagttcatc
      481 tgtactactg gtaaactgcc ggttccttgg ccgactctgg taacgacgct gacttatggt
      541 gttcagtgct ttgctcgtta tccggaccat atgaagcagc atgacttctt caagtccgcc
      601 atgccggaag gctatgtgca ggaacgcacg atttccttta aggatgacgg cacgtacaaa
      661 acgcgtgcgg aagtgaaatt tgaaggcgat accctggtaa accgcattga gctgaaaggc
      721 attgacttta aagaggacgg caatatcctg ggccataagc tggaatacaa ttttaacagc
      781 cacaatgttt acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa ttttaaaatt
      841 cgccacaacg tggaggatgg cagcgtgcag ctggctgatc actaccagca aaacactcca
      901 atcggtgatg gtcctgttct gctgccagac aatcactatc tgagcacgca aagcgttctg
      961 tctaaagatc cgaacgagaa acgcgatcat atggttctgc tggagttcgt aaccgcagcg
     1021 ggcatcacgc atggtatgga tgaactgtac aaatgaccag gcatcaaata aaacgaaagg
     1081 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact
     1141 agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatagctgg aaatctgctc
     1201 gtcagtggtg ctcacactga cgaatcatgt acagatcata ccgatgactg cctggcgact
     1261 cacaactaag caagacagcc ggaaccagcg ccggcgaaca ccactgcata tatggcatat
     1321 cacaacagtc cacgtctcaa gcagttacag agatgttacg aaccactagt gcactgcaga
     1381 tcgaattcga gttggcgcgc ctgtcattct aaatctctct ttcagcctaa agctttttcc
     1441 ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg
     1501 atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg
     1561 gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga
     1621 gggacgtaat tacatccctg ggggctttgg gagggggctg tccccgtgag ctcccaggcg
     1681 cgcctgtcat tctaaatctc tctttcagcc taaagctttt tccccgtatc cccccaggtg
     1741 tctgcaggct caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc
     1801 ccgtgcccgg gctgtccccg cacgctgccg gctcggggat gcggggggag cgccggaccg
     1861 gagcggagcc ccgggcggct cgctgctgcc ccctagcggg ggagggacgt aattacatcc
     1921 ctgggggctt tggggggggg ctgtccccgt gagctcaata aaagagccca ctacagtgcc
     1981 agggcgtgcc cttgggctcc ccgggcgcgg agtatgagcg agctgattaa ggagaacatg
     2041 cacatgaagc tgtacatgga gggcaccgtg gacaaccatc acttcaagtg cacatccgag
     2101 ggcgaaggca agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc
     2161 cctctcccct tcgccttcga catcctggct actagcttcc tctacggcag caagaccttc
     2221 atcaaccaca cccagggcat ccccgacttc ttcaagcagt ccttccctga gggcttcaca
     2281 tgggagagag tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc
     2341 ctccaggacg gctgcctcat ctacaacgtc aagatcagag gggtgaactt cacatccaac
     2401 ggccctgtga tgcagaagaa aacactcggc tgggaggcct tcaccgaaac gctgtacccc
     2461 gctgacggcg gcctggaagg cagaaacgac atggccctga agctcgtggg cgggagccat
     2521 ctgatcgcaa acatcaagac cacatataga tccaagaaac ccgctaagaa cctcaagatg
     2581 cctggcgtct actatgtgga ctacagactg gaaagaatca aggaggccaa caacgaaacc
     2641 tacgtcgagc agcacgaggt ggcagtggcc agatactgcg acctccctag caaactgggg
     2701 cacaagctta atcccaagaa gaagaggaag gtggaggacg cccgtacgag tactggaagc
     2761 ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggacctgga
     2821 tccatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca
     2881 atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac
     2941 ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc
     3001 gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc
     3061 ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat
     3121 ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg
     3181 attcgtgaat tgctgccctc tggttatgtg tgggagggct aactcgagcg acgcctcgac
     3241 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3301 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3361 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3421 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atccgagcgg ccgcgtgtta
     3481 caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     3541 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     3601 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     3661 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     3721 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt
     3781 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     3841 accgttattc attcgtgatt gcgcctgagc gaggcgaaat acgcgatcgc tgttaaaagg
     3901 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     3961 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     4021 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     4081 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     4141 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     4201 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     4261 catgttggaa tttaatcgcg gcctggagca agacgtttcc cgttgaatat ggctcataac
     4321 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt
     4381 atcttgtgca atgtaacatc agagattttg agacacaacg tggctttgtt gaataaatcg
     4441 aacttttgct gagttgaagg atcagtcatg accaaaatcc cttaacgtga gttttcgttc
     4501 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4561 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4621 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4681 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4741 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4801 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4861 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4921 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4981 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5041 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5101 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5161 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5221 gataaccgtg cggccgccaa tataacttcg tataatgtat gctatacgaa gttatccct
//
