LOCUS       sequence-271968-        5007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the spacer TU with connectors L3 R4 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_021
FEATURES             Location/Qualifiers
     source          1..5007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(19..37)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(join(144..935,936..1004))
                     /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     767..786
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1005..1109)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1212..1800
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1701..1720
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1912..1931
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1912..1930
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   1960..1994
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        3048..3251
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3202..3222
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3273..3971
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3972..3992
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4023..4076
                     /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             4083..4481
                     /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(4493..4510)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4499..4706
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4913..4975
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 ttaatccagc ttttgttccc tttagtgagg gttaattccg agcggccgcg attatcaaaa
       61 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
      121 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
      181 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
      241 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgggaccc acgctcaccg
      301 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
      361 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
      421 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
      481 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
      541 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
      601 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
      661 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
      721 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
      781 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
      841 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
      901 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
      961 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     1021 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     1081 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgtcatg
     1141 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     1201 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     1261 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     1321 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     1381 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1441 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1501 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1561 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     1621 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     1681 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     1741 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     1801 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     1861 ttctttcctg cgttatcccc tgattctgtg gataaccgtg cggccgccaa ttaatacgac
     1921 tcactatagg gcgaattggg gcgcgccatt ctagattaac cctagaaaga tagtctgcgt
     1981 aaaattgacg catgcattct tgaaatattg ctctctcttt ctaaatagcg cgaatccgtc
     2041 gctgtgcatt taggacatct cagtcgccgc ttggagctcc cgtgaggcgt gcttgtcaat
     2101 gcggtaagtg tcactgattt tgaactataa cgaccgcgtg agtcaaaatg acgcatgatt
     2161 atcttttacg tgacttttaa gatttaactc atacgataat tatattgtta tttcatgttc
     2221 tacttacgtg ataacttatt atatatatat tttcttgtta tagatatcaa ctagaatgct
     2281 agcatgggcc cagatctcca tgggccctga attcgcatct agagcagacc tagaccacat
     2341 gaggctgatg taggacagcc accagtggca gctaatcaac accgcaagat gccgatgcac
     2401 gctcatatca tcgtcgtcag cctggtagcc attcgacata cggatcaggg aactcgaacc
     2461 cagtaccgtc tcagttctgt atgggcacag acaacctaaa cgactagaga tctatgtgag
     2521 gatcctaact cgagatcgag ctgggtatga gacggcctgc actagacgaa ctaggcaaga
     2581 tgcgtccaat ccgtctaaac atggtgacaa cgctggacag atgacgtaac accgagccac
     2641 atcctgaaat cgaggcaggc taaccgaaac cgtgacaatg caaagagaca gcctgacact
     2701 agtgcactgc agtacagcgg tgggctctat ggggtaccta cgtaggtaca taacttacgg
     2761 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     2821 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     2881 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     2941 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     3001 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     3061 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     3121 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     3181 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     3241 taagcagagc tcgtttagtg aaccgtcaga taatgagcga gctgattaag gagaacatgc
     3301 acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg
     3361 gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc
     3421 ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca
     3481 tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat
     3541 gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc
     3601 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg
     3661 gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgaaacg ctgtaccccg
     3721 ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc
     3781 tgatcgcaaa catcaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc
     3841 ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgaaacct
     3901 acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc
     3961 acaagcttaa tcccaagaag aagaggaagg tggaggacgc ccgtacgagt actggaagcg
     4021 gagagggcag aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggacctggat
     4081 ccatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa
     4141 tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg
     4201 gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt gcagaactcg
     4261 tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg
     4321 gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg cttctcgatc
     4381 tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg gcagttggga
     4441 ttcgtgaatt gctgccctct ggttatgtgt gggagggcta actcgagcga cgcctcgact
     4501 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     4561 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     4621 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     4681 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tgagtatcga tactagttaa
     4741 aagttttgtt actttataga agaaattttg agtttttgtt tttttttaat aaataaataa
     4801 acataaataa attgtttgtt gaatttatta ttagtatgta agtgtaaata taataaaact
     4861 taatatctat tcaaattaat aaataaacct cgatatacag accgataaaa cacatgcgtc
     4921 aattttacgc atgattatct ttaacgtacg tcacaatatg attatctttc tagggttaat
     4981 ctagtatacg cgtatgcggc cgcttaa
//
