LOCUS       sequence-250106-        5881 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the piggyBac GFP dropout destination vector with the A1 
            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 5881)
  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 5881)
  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_043
FEATURES             Location/Qualifiers
     source          1..5881
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             322..333
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             340..1053
                     /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      1065..1136
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1152..1179
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        1883..2086
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2037..2057
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2108..2117
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2114..3142
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2114..3142
                     /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(3154..3171)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3160..3367
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3574..3636
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(3687..3705)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(3745..4560)
                     /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(3745..4560)
                     /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     4468..4487
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4552..4571
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4785..5373
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5274..5293
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5485..5504
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5485..5503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   5533..5567
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 gaattcgcat ctagactgat gagacgtggt agagccacaa acagccggta caagcaacga
       61 tctccaggac catctgaatc atgcgcggat gacacgaact cacgacggcg atcacagaca
      121 ttaacccaca gtacagacac tgcgacaacg tggcaattcg tcgcaataca acggaaagtg
      181 aaacgtgatt tcatgcgtca ttttgaacat tttgtaaatc ttatttaata atgtgtgcgg
      241 caattcacat ttaatttatg aatgttttct taacatcgcg gcaactcaag aaacggcagg
      301 ttcggatctt agctactaga gaaagaggag aaatactaga tgcgtaaagg cgaagagctg
      361 ttcactggtg tcgtccctat tctggtggaa ctggatggtg atgtcaacgg tcataagttt
      421 tccgtgcgtg gcgagggtga aggtgacgca actaatggta aactgacgct gaagttcatc
      481 tgtactactg gtaaactgcc ggttccttgg ccgactctgg taacgacgct gacttatggt
      541 gttcagtgct ttgctcgtta tccggaccat atgaagcagc atgacttctt caagtccgcc
      601 atgccggaag gctatgtgca ggaacgcacg atttccttta aggatgacgg cacgtacaaa
      661 acgcgtgcgg aagtgaaatt tgaaggcgat accctggtaa accgcattga gctgaaaggc
      721 attgacttta aagaggacgg caatatcctg ggccataagc tggaatacaa ttttaacagc
      781 cacaatgttt acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa ttttaaaatt
      841 cgccacaacg tggaggatgg cagcgtgcag ctggctgatc actaccagca aaacactcca
      901 atcggtgatg gtcctgttct gctgccagac aatcactatc tgagcacgca aagcgttctg
      961 tctaaagatc cgaacgagaa acgcgatcat atggttctgc tggagttcgt aaccgcagcg
     1021 ggcatcacgc atggtatgga tgaactgtac aaatgaccag gcatcaaata aaacgaaagg
     1081 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact
     1141 agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatagctgg aaatctgctc
     1201 gtcagtggtg ctcacactga cgaatcatgt acagatcata ccgatgactg cctggcgact
     1261 cacaactaag caagacagcc ggaaccagcg ccggcgaaca ccactgcata tatggcatat
     1321 cacaacagtc cacgtctcaa gcagttacag agatgttacg aaccctgaac aggttgacta
     1381 ttaattgtgt ctgcttgatg tggacaccag gtggcgctgg acatcagatt tggagaggca
     1441 gttgtctagg gaaccgggct ctgtgccagc gcaggaggca ggctggctct cctattccag
     1501 ggatgctcat ccaggaagga aaggttgcat gctggacaca ctaaccttac agcggtgggc
     1561 tctatggggt acctacgtag gtacataact tacggtaaat ggcccgcctg gctgaccgcc
     1621 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     1681 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     1741 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     1801 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     1861 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
     1921 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     1981 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     2041 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg
     2101 tcagatagcc accatgggta aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     2161 gatcgaaaag ttcgacagcg tgtccgacct gatgcagctc tcggagggcg aagaatctcg
     2221 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     2281 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
     2341 ggaagtgctt gacattgggg aatttagcga gagcctgacc tattgcatct cccgccgtgc
     2401 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcaaccggt
     2461 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     2521 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
     2581 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     2641 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     2701 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     2761 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     2821 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     2881 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     2941 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     3001 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     3061 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     3121 tcgtccgagg gcaaaggaat aactcgagcg acgcctcgac tgtgccttct agttgccagc
     3181 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3241 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3301 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3361 ctggggatgc ggtgggctct atgagtatcg atactagtta aaagttttgt tactttatag
     3421 aagaaatttt gagtttttgt ttttttttaa taaataaata aacataaata aattgtttgt
     3481 tgaatttatt attagtatgt aagtgtaaat ataataaaac ttaatatcta ttcaaattaa
     3541 taaataaacc tcgatataca gaccgataaa acacatgcgt caattttacg catgattatc
     3601 tttaacgtac gtcacaatat gattatcttt ctagggttaa tctagtatac gcgtatgcgg
     3661 ccgcttaatt aatccagctt ttgttccctt tagtgagggt taattccgag cggccgcgtg
     3721 ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat gaaactgcaa
     3781 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     3841 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     3901 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     3961 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct tatgcatttc
     4021 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     4081 caaaccgtta ttcattcgtg attgcgcctg agcgaggcga aatacgcgat cgctgttaaa
     4141 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     4201 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     4261 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     4321 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     4381 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     4441 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     4501 atccatgttg gaatttaatc gcggcctgga gcaagacgtt tcccgttgaa tatggctcat
     4561 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt
     4621 tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtggcttt gttgaataaa
     4681 tcgaactttt gctgagttga aggatcagtc atgaccaaaa tcccttaacg tgagttttcg
     4741 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4801 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4861 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4921 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4981 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     5041 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     5101 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     5161 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     5221 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     5281 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5341 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5401 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     5461 gtggataacc gtgcggccgc caattaatac gactcactat agggcgaatt ggggcgcgcc
     5521 attctagatt aaccctagaa agatagtctg cgtaaaattg acgcatgcat tcttgaaata
     5581 ttgctctctc tttctaaata gcgcgaatcc gtcgctgtgc atttaggaca tctcagtcgc
     5641 cgcttggagc tcccgtgagg cgtgcttgtc aatgcggtaa gtgtcactga ttttgaacta
     5701 taacgaccgc gtgagtcaaa atgacgcatg attatctttt acgtgacttt taagatttaa
     5761 ctcatacgat aattatattg ttatttcatg ttctacttac gtgataactt attatatata
     5821 tattttcttg ttatagatat caactagaat gctagcatgg gcccagatct ccatgggccc
     5881 t
//
