LOCUS       sequence-250082-        6479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the piggyBac GFP dropout destination vector with the 2xHS4 
            insulator and 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 6479)
  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 6479)
  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_015
FEATURES             Location/Qualifiers
     source          1..6479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             606..1304
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             1305..1325
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1356..1409
                     /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             1416..1814
                     /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(1826..1843)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1832..2039
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2246..2308
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(2359..2377)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(2417..3232)
                     /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(2417..3232)
                     /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     3140..3159
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3224..3243
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3457..4045
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3946..3965
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4157..4176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4157..4175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   4205..4239
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     RBS             4875..4886
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4893..5606
                     /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      5618..5689
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5705..5732
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
ORIGIN
        1 gctttggggg ggggctgtcc ccgtgagctc aataaaagag cccactacag cggtgggctc
       61 tatggggtac ctacgtaggt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agataatgag cgagctgatt aaggagaaca tgcacatgaa gctgtacatg gagggcaccg
      661 tggacaacca tcacttcaag tgcacatccg agggcgaagg caagccctac gagggcaccc
      721 agaccatgag aatcaaggtg gtcgagggcg gccctctccc cttcgccttc gacatcctgg
      781 ctactagctt cctctacggc agcaagacct tcatcaacca cacccagggc atccccgact
      841 tcttcaagca gtccttccct gagggcttca catgggagag agtcaccaca tacgaagacg
      901 ggggcgtgct gaccgctacc caggacacca gcctccagga cggctgcctc atctacaacg
      961 tcaagatcag aggggtgaac ttcacatcca acggccctgt gatgcagaag aaaacactcg
     1021 gctgggaggc cttcaccgaa acgctgtacc ccgctgacgg cggcctggaa ggcagaaacg
     1081 acatggccct gaagctcgtg ggcgggagcc atctgatcgc aaacatcaag accacatata
     1141 gatccaagaa acccgctaag aacctcaaga tgcctggcgt ctactatgtg gactacagac
     1201 tggaaagaat caaggaggcc aacaacgaaa cctacgtcga gcagcacgag gtggcagtgg
     1261 ccagatactg cgacctccct agcaaactgg ggcacaagct taatcccaag aagaagagga
     1321 aggtggagga cgcccgtacg agtactggaa gcggagaggg cagaggaagt ctgctaacat
     1381 gcggtgacgt cgaggagaat cctggacctg gatccatggc caagcctttg tctcaagaag
     1441 aatccaccct cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact
     1501 acagcgtcgc cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat
     1561 atcattttac tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg
     1621 cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc
     1681 cctgcggacg gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga
     1741 aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg
     1801 tgtgggaggg ctaactcgag cgacgcctcg actgtgcctt ctagttgcca gccatctgtt
     1861 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     1921 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     1981 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2041 gcggtgggct ctatgagtat cgatactagt taaaagtttt gttactttat agaagaaatt
     2101 ttgagttttt gttttttttt aataaataaa taaacataaa taaattgttt gttgaattta
     2161 ttattagtat gtaagtgtaa atataataaa acttaatatc tattcaaatt aataaataaa
     2221 cctcgatata cagaccgata aaacacatgc gtcaatttta cgcatgatta tctttaacgt
     2281 acgtcacaat atgattatct ttctagggtt aatctagtat acgcgtatgc ggccgcttaa
     2341 ttaatccagc ttttgttccc tttagtgagg gttaattccg agcggccgcg tgttacaacc
     2401 aattaaccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2461 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     2521 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     2581 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     2641 caccatgagt gacgactgaa tccggtgaga atggcaaaag cttatgcatt tctttccaga
     2701 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2761 tattcattcg tgattgcgcc tgagcgaggc gaaatacgcg atcgctgtta aaaggacaat
     2821 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2881 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     2941 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     3001 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     3061 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     3121 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     3181 tggaatttaa tcgcggcctg gagcaagacg tttcccgttg aatatggctc ataacacccc
     3241 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt
     3301 gtgcaatgta acatcagaga ttttgagaca caacgtggct ttgttgaata aatcgaactt
     3361 ttgctgagtt gaaggatcag tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3421 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3481 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3541 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3601 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3661 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3721 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3781 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3841 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3901 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3961 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4021 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4081 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4141 ccgtgcggcc gccaattaat acgactcact atagggcgaa ttggggcgcg ccattctaga
     4201 ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc
     4261 tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga
     4321 gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc
     4381 gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg
     4441 ataattatat tgttatttca tgttctactt acgtgataac ttattatata tatattttct
     4501 tgttatagat atcaactaga atgctagcat gggcccagat ctccatgggc cctgaattcg
     4561 catctagact gatgagacgt ggtagagcca caaacagccg gtacaagcaa cgatctccag
     4621 gaccatctga atcatgcgcg gatgacacga actcacgacg gcgatcacag acattaaccc
     4681 acagtacaga cactgcgaca acgtggcaat tcgtcgcaat acaacggaaa gtgaaacgtg
     4741 atttcatgcg tcattttgaa cattttgtaa atcttattta ataatgtgtg cggcaattca
     4801 catttaattt atgaatgttt tcttaacatc gcggcaactc aagaaacggc aggttcggat
     4861 cttagctact agagaaagag gagaaatact agatgcgtaa aggcgaagag ctgttcactg
     4921 gtgtcgtccc tattctggtg gaactggatg gtgatgtcaa cggtcataag ttttccgtgc
     4981 gtggcgaggg tgaaggtgac gcaactaatg gtaaactgac gctgaagttc atctgtacta
     5041 ctggtaaact gccggttcct tggccgactc tggtaacgac gctgacttat ggtgttcagt
     5101 gctttgctcg ttatccggac catatgaagc agcatgactt cttcaagtcc gccatgccgg
     5161 aaggctatgt gcaggaacgc acgatttcct ttaaggatga cggcacgtac aaaacgcgtg
     5221 cggaagtgaa atttgaaggc gataccctgg taaaccgcat tgagctgaaa ggcattgact
     5281 ttaaagagga cggcaatatc ctgggccata agctggaata caattttaac agccacaatg
     5341 tttacatcac cgccgataaa caaaaaaatg gcattaaagc gaattttaaa attcgccaca
     5401 acgtggagga tggcagcgtg cagctggctg atcactacca gcaaaacact ccaatcggtg
     5461 atggtcctgt tctgctgcca gacaatcact atctgagcac gcaaagcgtt ctgtctaaag
     5521 atccgaacga gaaacgcgat catatggttc tgctggagtt cgtaaccgca gcgggcatca
     5581 cgcatggtat ggatgaactg tacaaatgac caggcatcaa ataaaacgaa aggctcagtc
     5641 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca
     5701 cactggctca ccttcgggtg ggcctttctg cgtttatagc tggaaatctg ctcgtcagtg
     5761 gtgctcacac tgacgaatca tgtacagatc ataccgatga ctgcctggcg actcacaact
     5821 aagcaagaca gccggaacca gcgccggcga acaccactgc atatatggca tatcacaaca
     5881 gtccacgtct caagcagtta cagagatgtt acgaaccact agtgcactgc agatcgaatt
     5941 cgagttggcg cgcctgtcat tctaaatctc tctttcagcc taaagctttt tccccgtatc
     6001 cccccaggtg tctgcaggct caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt
     6061 gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg gctcggggat gcggggggag
     6121 cgccggaccg gagcggagcc ccgggcggct cgctgctgcc ccctagcggg ggagggacgt
     6181 aattacatcc ctgggggctt tgggaggggg ctgtccccgt gagctcccag gcgcgcctgt
     6241 cattctaaat ctctctttca gcctaaagct ttttccccgt atccccccag gtgtctgcag
     6301 gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc
     6361 cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga ccggagcgga
     6421 gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca tccctgggg
//
