LOCUS       sequence-250087-        5007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the spacer TU with connectors LS R1 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_018
FEATURES             Location/Qualifiers
     source          1..5007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     312..331
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(550..654)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      757..1345
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1246..1265
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1457..1476
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1457..1475
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   1505..1539
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        2593..2796
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2747..2767
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2818..3516
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3517..3537
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3568..3621
                     /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             3628..4026
                     /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(4038..4055)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4044..4251
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4458..4520
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(4571..4589)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(join(4696..5007,1..480,481..549))
                     /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"
ORIGIN
        1 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
       61 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
      121 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
      181 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
      241 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
      301 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
      361 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
      421 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
      481 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
      541 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
      601 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
      661 tccccgaaaa gtgccacctg tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
      721 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
      781 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
      841 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
      901 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
      961 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     1021 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     1081 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     1141 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     1201 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     1261 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     1321 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     1381 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     1441 ccgtgcggcc gccaattaat acgactcact atagggcgaa ttggggcgcg ccattctaga
     1501 ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc
     1561 tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga catctcagtc gccgcttgga
     1621 gctcccgtga ggcgtgcttg tcaatgcggt aagtgtcact gattttgaac tataacgacc
     1681 gcgtgagtca aaatgacgca tgattatctt ttacgtgact tttaagattt aactcatacg
     1741 ataattatat tgttatttca tgttctactt acgtgataac ttattatata tatattttct
     1801 tgttatagat atcaactaga atgctagcat gggcccagat ctccatgggc cctgaattcg
     1861 catctagatg gtagagccac aaacagccgg tacaagcaac gatctccagg accatctgaa
     1921 tcatgcgcgg atgacacgaa ctcacgacgg cgatcacaga cattaaccca cagtacagac
     1981 actgcgacaa cgtggcaatt cgtcgcaata ccgtctcact gaactggccg ataattgcag
     2041 acgaacgact agagatctat gtgaggatcc taactcgaga tcgagctgcc aatgagacga
     2101 cggggtcatc acggctcatc atgcgccaaa caaatgtgtg caatacacgc tcggatgact
     2161 gcatgatgac cgcactgact ggggacagca gatccaccta agcctgtgag agaagcagac
     2221 acccgacaga tcaaggcagt taactagtgc actgcagtac agcggtgggc tctatggggt
     2281 acctacgtag gtacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     2341 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     2401 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     2461 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     2521 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     2581 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     2641 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     2701 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     2761 aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg tcagataatg
     2821 agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac cgtggacaac
     2881 catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg
     2941 agaatcaagg tggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctactagc
     3001 ttcctctacg gcagcaagac cttcatcaac cacacccagg gcatccccga cttcttcaag
     3061 cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga cgggggcgtg
     3121 ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc
     3181 agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact cggctgggag
     3241 gccttcaccg aaacgctgta ccccgctgac ggcggcctgg aaggcagaaa cgacatggcc
     3301 ctgaagctcg tgggcgggag ccatctgatc gcaaacatca agaccacata tagatccaag
     3361 aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag actggaaaga
     3421 atcaaggagg ccaacaacga aacctacgtc gagcagcacg aggtggcagt ggccagatac
     3481 tgcgacctcc ctagcaaact ggggcacaag cttaatccca agaagaagag gaaggtggag
     3541 gacgcccgta cgagtactgg aagcggagag ggcagaggaa gtctgctaac atgcggtgac
     3601 gtcgaggaga atcctggacc tggatccatg gccaagcctt tgtctcaaga agaatccacc
     3661 ctcattgaaa gagcaacggc tacaatcaac agcatcccca tctctgaaga ctacagcgtc
     3721 gccagcgcag ctctctctag cgacggccgc atcttcactg gtgtcaatgt atatcatttt
     3781 actgggggac cttgtgcaga actcgtggtg ctgggcactg ctgctgctgc ggcagctggc
     3841 aacctgactt gtatcgtcgc gatcggaaat gagaacaggg gcatcttgag cccctgcgga
     3901 cggtgccgac aggtgcttct cgatctgcat cctgggatca aagccatagt gaaggacagt
     3961 gatggacagc cgacggcagt tgggattcgt gaattgctgc cctctggtta tgtgtgggag
     4021 ggctaactcg agcgacgcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     4081 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4141 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4201 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     4261 ctctatgagt atcgatacta gttaaaagtt ttgttacttt atagaagaaa ttttgagttt
     4321 ttgttttttt ttaataaata aataaacata aataaattgt ttgttgaatt tattattagt
     4381 atgtaagtgt aaatataata aaacttaata tctattcaaa ttaataaata aacctcgata
     4441 tacagaccga taaaacacat gcgtcaattt tacgcatgat tatctttaac gtacgtcaca
     4501 atatgattat ctttctaggg ttaatctagt atacgcgtat gcggccgctt aattaatcca
     4561 gcttttgttc cctttagtga gggttaattc cgagcggccg cgattatcaa aaaggatctt
     4621 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4681 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4741 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4801 cttaccatct ggccccagtg ctgcaatgat accgcgggac ccacgctcac cggctccaga
     4861 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4921 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4981 taatagtttg cgcaacgttg ttgccat
//
