LOCUS       sequence-250097-        5007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the spacer TU with connectors L2 R3 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 5007)
  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 5007)
  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_020
FEATURES             Location/Qualifiers
     source          1..5007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(18..36)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(join(143..934,935..1003))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     766..785
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1004..1108)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1211..1799
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1700..1719
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1911..1930
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1911..1929
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   1959..1993
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        3047..3250
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3201..3221
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3272..3970
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3971..3991
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4022..4075
                     /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             4082..4480
                     /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(4492..4509)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4498..4705
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4912..4974
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 taatccagct tttgttccct ttagtgaggg ttaattccga gcggccgcga ttatcaaaaa
       61 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
      121 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
      181 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
      241 gggagggctt accatctggc cccagtgctg caatgatacc gcgggaccca cgctcaccgg
      301 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
      361 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
      421 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
      481 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
      541 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
      601 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
      661 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
      721 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
      781 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
      841 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
      901 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
      961 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     1021 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     1081 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgtcatga
     1141 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     1201 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     1261 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     1321 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     1381 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     1441 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     1501 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     1561 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     1621 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     1681 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     1741 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     1801 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     1861 tctttcctgc gttatcccct gattctgtgg ataaccgtgc ggccgccaat taatacgact
     1921 cactataggg cgaattgggg cgcgccattc tagattaacc ctagaaagat agtctgcgta
     1981 aaattgacgc atgcattctt gaaatattgc tctctctttc taaatagcgc gaatccgtcg
     2041 ctgtgcattt aggacatctc agtcgccgct tggagctccc gtgaggcgtg cttgtcaatg
     2101 cggtaagtgt cactgatttt gaactataac gaccgcgtga gtcaaaatga cgcatgatta
     2161 tcttttacgt gacttttaag atttaactca tacgataatt atattgttat ttcatgttct
     2221 acttacgtga taacttatta tatatatatt ttcttgttat agatatcaac tagaatgcta
     2281 gcatgggccc agatctccat gggccctgaa ttcgcatcta gaataaatcg cagccaagtg
     2341 agtgaataga tgacgcacca cggtcagaca cggccacatc gtatctcaca ggagcaagcg
     2401 cgataggagc actcacacat agtacggtga tccgctgact cctttgccca aataagacgt
     2461 gagcccgtct cagatgcaca cactggctta agatgacaac gactagagat ctatgtgagg
     2521 atcctaactc gagatcgagc tggttctgag acggcagacc tagaccacat gaggctgatg
     2581 taggacagcc accagtggca gctaatcaac accgcaagat gccgatgcac gctcatatca
     2641 tcgtcgtcag cctggtagcc attcgacata cggatcaggg aactcgaacc cagtacacta
     2701 gtgcactgca gtacagcggt gggctctatg gggtacctac gtaggtacat aacttacggt
     2761 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     2821 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     2881 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     2941 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     3001 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     3061 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     3121 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     3181 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     3241 aagcagagct cgtttagtga accgtcagat aatgagcgag ctgattaagg agaacatgca
     3301 catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca catccgaggg
     3361 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
     3421 tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca agaccttcat
     3481 caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg gcttcacatg
     3541 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct
     3601 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca catccaacgg
     3661 ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgaaacgc tgtaccccgc
     3721 tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg ggagccatct
     3781 gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc tcaagatgcc
     3841 tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca acgaaaccta
     3901 cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca aactggggca
     3961 caagcttaat cccaagaaga agaggaaggt ggaggacgcc cgtacgagta ctggaagcgg
     4021 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gacctggatc
     4081 catggccaag cctttgtctc aagaagaatc caccctcatt gaaagagcaa cggctacaat
     4141 caacagcatc cccatctctg aagactacag cgtcgccagc gcagctctct ctagcgacgg
     4201 ccgcatcttc actggtgtca atgtatatca ttttactggg ggaccttgtg cagaactcgt
     4261 ggtgctgggc actgctgctg ctgcggcagc tggcaacctg acttgtatcg tcgcgatcgg
     4321 aaatgagaac aggggcatct tgagcccctg cggacggtgc cgacaggtgc ttctcgatct
     4381 gcatcctggg atcaaagcca tagtgaagga cagtgatgga cagccgacgg cagttgggat
     4441 tcgtgaattg ctgccctctg gttatgtgtg ggagggctaa ctcgagcgac gcctcgactg
     4501 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     4561 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     4621 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     4681 aagacaatag caggcatgct ggggatgcgg tgggctctat gagtatcgat actagttaaa
     4741 agttttgtta ctttatagaa gaaattttga gtttttgttt ttttttaata aataaataaa
     4801 cataaataaa ttgtttgttg aatttattat tagtatgtaa gtgtaaatat aataaaactt
     4861 aatatctatt caaattaata aataaacctc gatatacaga ccgataaaac acatgcgtca
     4921 attttacgca tgattatctt taacgtacgt cacaatatga ttatctttct agggttaatc
     4981 tagtatacgc gtatgcggcc gcttaat
//
