LOCUS       sequence-251652-        6319 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Encodes the piggyBac GFP dropout destination vector with the 2xHS4 
            insulator 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 6319)
  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 6319)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_002
FEATURES             Location/Qualifiers
     source          1..6319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      184..772
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     673..692
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     884..903
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        884..902
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   932..966
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     RBS             1609..1620
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1627..2340
                     /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      2352..2423
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2439..2466
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        3601..3804
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3755..3775
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3826..3835
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3832..4860
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             3832..4860
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     primer_bind     complement(4872..4889)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4878..5085
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5292..5354
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(5405..5423)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(5463..6278)
                     /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(5463..6278)
                     /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     6186..6205
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6270..6289
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa
       61 cgtggctttg ttgaataaat cgaacttttg ctgagttgaa ggatcagtca tgaccaaaat
      121 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
      181 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
      241 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
      301 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
      361 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
      421 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
      481 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
      541 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
      601 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      661 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      721 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      781 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      841 tgcgttatcc cctgattctg tggataaccg tgcggccgcc aattaatacg actcactata
      901 gggcgaattg gggcgcgcca ttctagatta accctagaaa gatagtctgc gtaaaattga
      961 cgcatgcatt cttgaaatat tgctctctct ttctaaatag cgcgaatccg tcgctgtgca
     1021 tttaggacat ctcagtcgcc gcttggagct cccgtgaggc gtgcttgtca atgcggtaag
     1081 tgtcactgat tttgaactat aacgaccgcg tgagtcaaaa tgacgcatga ttatctttta
     1141 cgtgactttt aagatttaac tcatacgata attatattgt tatttcatgt tctacttacg
     1201 tgataactta ttatatatat attttcttgt tatagatatc aactagaatg ctagcatggg
     1261 cccagatctc catgggccct gaattcgcat ctagactgat gagacgtggt agagccacaa
     1321 acagccggta caagcaacga tctccaggac catctgaatc atgcgcggat gacacgaact
     1381 cacgacggcg atcacagaca ttaacccaca gtacagacac tgcgacaacg tggcaattcg
     1441 tcgcaataca acgtgagacc gaaagtgaaa cgtgatttca tgcgtcattt tgaacatttt
     1501 gtaaatctta tttaataatg tgtgcggcaa ttcacattta atttatgaat gttttcttaa
     1561 catcgcggca actcaagaaa cggcaggttc ggatcttagc tactagagaa agaggagaaa
     1621 tactagatgc gtaaaggcga agagctgttc actggtgtcg tccctattct ggtggaactg
     1681 gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg tgacgcaact
     1741 aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt tccttggccg
     1801 actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc ggaccatatg
     1861 aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga acgcacgatt
     1921 tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga aggcgatacc
     1981 ctggtaaacc gcattgagct gaaaggcatt gactttaaag aggacggcaa tatcctgggc
     2041 cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga taaacaaaaa
     2101 aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag cgtgcagctg
     2161 gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct gccagacaat
     2221 cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg cgatcatatg
     2281 gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gtatggatga actgtacaaa
     2341 tgaccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     2401 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt
     2461 tctgcgttta taggtctcag ctggaaatct gctcgtcagt ggtgctcaca ctgacgaatc
     2521 atgtacagat cataccgatg actgcctggc gactcacaac taagcaagac agccggaacc
     2581 agcgccggcg aacaccactg catatatggc atatcacaac agtccacgtc tcaagcagtt
     2641 acagagatgt tacgaaccac tagtgcactg cagatcgaat tcgagttggc gcgcctgtca
     2701 ttctaaatct ctctttcagc ctaaagcttt ttccccgtat ccccccaggt gtctgcaggc
     2761 tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg
     2821 ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc
     2881 cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct
     2941 ttgggagggg gctgtccccg tgagctccca ggcgcgcctg tcattctaaa tctctctttc
     3001 agcctaaagc tttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa
     3061 gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct
     3121 gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc
     3181 tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg ggggctgtcc
     3241 ccgtgagctc aataaaagag cccactacag cggtgggctc tatggggtac ctacgtaggt
     3301 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     3361 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     3421 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     3481 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     3541 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     3601 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     3661 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     3721 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     3781 tgggaggtct atataagcag agctcgttta gtgaaccgtc agatagccac catgggtaaa
     3841 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtg
     3901 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     3961 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     4021 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     4081 tttagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     4141 ctgcctgaaa ccgaactgcc cgctgttctg caaccggtcg cggaggccat ggatgcgatc
     4201 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     4261 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     4321 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     4381 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     4441 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     4501 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     4561 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     4621 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     4681 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     4741 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     4801 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaataa
     4861 ctcgagcgac gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     4921 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     4981 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     5041 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     5101 gagtatcgat actagttaaa agttttgtta ctttatagaa gaaattttga gtttttgttt
     5161 ttttttaata aataaataaa cataaataaa ttgtttgttg aatttattat tagtatgtaa
     5221 gtgtaaatat aataaaactt aatatctatt caaattaata aataaacctc gatatacaga
     5281 ccgataaaac acatgcgtca attttacgca tgattatctt taacgtacgt cacaatatga
     5341 ttatctttct agggttaatc tagtatacgc gtatgcggcc gcttaattaa tccagctttt
     5401 gttcccttta gtgagggtta attccgagcg gccgcgtgtt acaaccaatt aaccaattct
     5461 gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     5521 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     5581 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     5641 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     5701 actgaatccg gtgagaatgg caaaagctta tgcatttctt tccagacttg ttcaacaggc
     5761 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     5821 tgcgcctgag cgaggcgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     5881 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     5941 tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca
     6001 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     6061 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     6121 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     6181 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     6241 ggcctggagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     6301 atgtaagcag acagtttta
//
