LOCUS       sequence-250100-        5007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the spacer TU with connectors L4 R5 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_022
FEATURES             Location/Qualifiers
     source          1..5007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(17..35)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(join(142..933,934..1002))
                     /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     765..784
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1003..1107)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1210..1798
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1699..1718
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1910..1929
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1910..1928
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   1958..1992
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        3046..3249
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3200..3220
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3271..3969
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3970..3990
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4021..4074
                     /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             4081..4479
                     /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(4491..4508)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4497..4704
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4911..4973
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 aatccagctt ttgttccctt tagtgagggt taattccgag cggccgcgat tatcaaaaag
       61 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
      121 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
      181 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
      241 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgggacccac gctcaccggc
      301 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
      361 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
      421 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
      481 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
      541 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
      601 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
      661 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
      721 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
      781 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
      841 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
      901 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
      961 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     1021 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1081 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgtcatgac
     1141 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1201 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1261 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1321 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     1381 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1441 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     1501 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     1561 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     1621 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     1681 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     1741 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     1801 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     1861 ctttcctgcg ttatcccctg attctgtgga taaccgtgcg gccgccaatt aatacgactc
     1921 actatagggc gaattggggc gcgccattct agattaaccc tagaaagata gtctgcgtaa
     1981 aattgacgca tgcattcttg aaatattgct ctctctttct aaatagcgcg aatccgtcgc
     2041 tgtgcattta ggacatctca gtcgccgctt ggagctcccg tgaggcgtgc ttgtcaatgc
     2101 ggtaagtgtc actgattttg aactataacg accgcgtgag tcaaaatgac gcatgattat
     2161 cttttacgtg acttttaaga tttaactcat acgataatta tattgttatt tcatgttcta
     2221 cttacgtgat aacttattat atatatattt tcttgttata gatatcaact agaatgctag
     2281 catgggccca gatctccatg ggccctgaat tcgcatctag agcctgcact agacgaacta
     2341 ggcaagatgc gtccaatccg tctaaacatg gtgacaacgc tggacagatg acgtaacacc
     2401 gagccacatc ctgaaatcga ggcaggctaa ccgaaaccgt gacaatgcaa agagacagcc
     2461 tgaccgtctc aggtagacta cccatgagtc acaatgaacg actagagatc tatgtgagga
     2521 tcctaactcg agatcgagct gaagttgaga cgacgcacaa ggtcagggca ctcatgcgac
     2581 aatcaactcg atgcatgatc cgcaccattg tcgaggggcc agcgtcaata gtgccgatga
     2641 ccacagaccc ggttaagaca tagccgaatg gagccgcgcc gaccacagaa tgataactag
     2701 tgcactgcag tacagcggtg ggctctatgg ggtacctacg taggtacata acttacggta
     2761 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     2821 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     2881 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
     2941 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
     3001 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
     3061 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     3121 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     3181 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     3241 agcagagctc gtttagtgaa ccgtcagata atgagcgagc tgattaagga gaacatgcac
     3301 atgaagctgt acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc
     3361 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct
     3421 ctccccttcg ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc
     3481 aaccacaccc agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg
     3541 gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc
     3601 caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc
     3661 cctgtgatgc agaagaaaac actcggctgg gaggccttca ccgaaacgct gtaccccgct
     3721 gacggcggcc tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg
     3781 atcgcaaaca tcaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct
     3841 ggcgtctact atgtggacta cagactggaa agaatcaagg aggccaacaa cgaaacctac
     3901 gtcgagcagc acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac
     3961 aagcttaatc ccaagaagaa gaggaaggtg gaggacgccc gtacgagtac tggaagcgga
     4021 gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg acctggatcc
     4081 atggccaagc ctttgtctca agaagaatcc accctcattg aaagagcaac ggctacaatc
     4141 aacagcatcc ccatctctga agactacagc gtcgccagcg cagctctctc tagcgacggc
     4201 cgcatcttca ctggtgtcaa tgtatatcat tttactgggg gaccttgtgc agaactcgtg
     4261 gtgctgggca ctgctgctgc tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga
     4321 aatgagaaca ggggcatctt gagcccctgc ggacggtgcc gacaggtgct tctcgatctg
     4381 catcctggga tcaaagccat agtgaaggac agtgatggac agccgacggc agttgggatt
     4441 cgtgaattgc tgccctctgg ttatgtgtgg gagggctaac tcgagcgacg cctcgactgt
     4501 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     4561 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     4621 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     4681 agacaatagc aggcatgctg gggatgcggt gggctctatg agtatcgata ctagttaaaa
     4741 gttttgttac tttatagaag aaattttgag tttttgtttt tttttaataa ataaataaac
     4801 ataaataaat tgtttgttga atttattatt agtatgtaag tgtaaatata ataaaactta
     4861 atatctattc aaattaataa ataaacctcg atatacagac cgataaaaca catgcgtcaa
     4921 ttttacgcat gattatcttt aacgtacgtc acaatatgat tatctttcta gggttaatct
     4981 agtatacgcg tatgcggccg cttaatt
//
