LOCUS       sequence-250084-        5291 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the BxBI 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 5291)
  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 5291)
  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_017
FEATURES             Location/Qualifiers
     source          1..5291
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    13..46
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     RBS             372..383
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             390..1103
                     /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      1115..1186
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1202..1229
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    2030..2067
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             2076..2774
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             2775..2795
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2826..2879
                     /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             2886..3284
                     /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(3296..3313)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3302..3509
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(3564..4379)
                     /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(3564..4379)
                     /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     4287..4306
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4371..4390
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4604..5192
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5093..5112
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcggccgcca atataacttc gtataatgta tgctatacga agttatccct gaattcgcat
       61 ctagactgat gagacgtggt agagccacaa acagccggta caagcaacga tctccaggac
      121 catctgaatc atgcgcggat gacacgaact cacgacggcg atcacagaca ttaacccaca
      181 gtacagacac tgcgacaacg tggcaattcg tcgcaataca acggaaagtg aaacgtgatt
      241 tcatgcgtca ttttgaacat tttgtaaatc ttatttaata atgtgtgcgg caattcacat
      301 ttaatttatg aatgttttct taacatcgcg gcaactcaag aaacggcagg ttcggatctt
      361 agctactaga gaaagaggag aaatactaga tgcgtaaagg cgaagagctg ttcactggtg
      421 tcgtccctat tctggtggaa ctggatggtg atgtcaacgg tcataagttt tccgtgcgtg
      481 gcgagggtga aggtgacgca actaatggta aactgacgct gaagttcatc tgtactactg
      541 gtaaactgcc ggttccttgg ccgactctgg taacgacgct gacttatggt gttcagtgct
      601 ttgctcgtta tccggaccat atgaagcagc atgacttctt caagtccgcc atgccggaag
      661 gctatgtgca ggaacgcacg atttccttta aggatgacgg cacgtacaaa acgcgtgcgg
      721 aagtgaaatt tgaaggcgat accctggtaa accgcattga gctgaaaggc attgacttta
      781 aagaggacgg caatatcctg ggccataagc tggaatacaa ttttaacagc cacaatgttt
      841 acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa ttttaaaatt cgccacaacg
      901 tggaggatgg cagcgtgcag ctggctgatc actaccagca aaacactcca atcggtgatg
      961 gtcctgttct gctgccagac aatcactatc tgagcacgca aagcgttctg tctaaagatc
     1021 cgaacgagaa acgcgatcat atggttctgc tggagttcgt aaccgcagcg ggcatcacgc
     1081 atggtatgga tgaactgtac aaatgaccag gcatcaaata aaacgaaagg ctcagtcgaa
     1141 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac
     1201 tggctcacct tcgggtgggc ctttctgcgt ttatagctgg aaatctgctc gtcagtggtg
     1261 ctcacactga cgaatcatgt acagatcata ccgatgactg cctggcgact cacaactaag
     1321 caagacagcc ggaaccagcg ccggcgaaca ccactgcata tatggcatat cacaacagtc
     1381 cacgtctcaa gcagttacag agatgttacg aaccactagt gcactgcaga tcgaattcga
     1441 gttggcgcgc ctgtcattct aaatctctct ttcagcctaa agctttttcc ccgtatcccc
     1501 ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg atcccgtgcc
     1561 accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg gggggagcgc
     1621 cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga gggacgtaat
     1681 tacatccctg ggggctttgg gagggggctg tccccgtgag ctcccaggcg cgcctgtcat
     1741 tctaaatctc tctttcagcc taaagctttt tccccgtatc cccccaggtg tctgcaggct
     1801 caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg
     1861 gctgtccccg cacgctgccg gctcggggat gcggggggag cgccggaccg gagcggagcc
     1921 ccgggcggct cgctgctgcc ccctagcggg ggagggacgt aattacatcc ctgggggctt
     1981 tggggggggg ctgtccccgt gagctcaata aaagagccca ctacaggccg gcttgtcgac
     2041 gacggcggtc tccgtcgtca ggatcatccg ggagtatgag cgagctgatt aaggagaaca
     2101 tgcacatgaa gctgtacatg gagggcaccg tggacaacca tcacttcaag tgcacatccg
     2161 agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtg gtcgagggcg
     2221 gccctctccc cttcgccttc gacatcctgg ctactagctt cctctacggc agcaagacct
     2281 tcatcaacca cacccagggc atccccgact tcttcaagca gtccttccct gagggcttca
     2341 catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc caggacacca
     2401 gcctccagga cggctgcctc atctacaacg tcaagatcag aggggtgaac ttcacatcca
     2461 acggccctgt gatgcagaag aaaacactcg gctgggaggc cttcaccgaa acgctgtacc
     2521 ccgctgacgg cggcctggaa ggcagaaacg acatggccct gaagctcgtg ggcgggagcc
     2581 atctgatcgc aaacatcaag accacatata gatccaagaa acccgctaag aacctcaaga
     2641 tgcctggcgt ctactatgtg gactacagac tggaaagaat caaggaggcc aacaacgaaa
     2701 cctacgtcga gcagcacgag gtggcagtgg ccagatactg cgacctccct agcaaactgg
     2761 ggcacaagct taatcccaag aagaagagga aggtggagga cgcccgtacg agtactggaa
     2821 gcggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggacctg
     2881 gatccatggc caagcctttg tctcaagaag aatccaccct cattgaaaga gcaacggcta
     2941 caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct ctctctagcg
     3001 acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct tgtgcagaac
     3061 tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt atcgtcgcga
     3121 tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag gtgcttctcg
     3181 atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg acggcagttg
     3241 ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaactcgag cgacgcctcg
     3301 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     3361 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     3421 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     3481 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatccgagc ggccgcgtgt
     3541 tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg aaactgcaat
     3601 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     3661 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     3721 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     3781 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct
     3841 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc
     3901 aaaccgttat tcattcgtga ttgcgcctga gcgaggcgaa atacgcgatc gctgttaaaa
     3961 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     4021 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc
     4081 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     4141 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg
     4201 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     4261 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     4321 tccatgttgg aatttaatcg cggcctggag caagacgttt cccgttgaat atggctcata
     4381 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt
     4441 ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttg ttgaataaat
     4501 cgaacttttg ctgagttgaa ggatcagtca tgaccaaaat cccttaacgt gagttttcgt
     4561 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4621 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4681 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4741 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4801 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4861 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4921 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4981 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5041 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5101 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5161 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5221 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5281 tggataaccg t
//
