LOCUS       sequence-250127-        5299 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the hAAVS1 homology landing pad with GFP dropout destination
            vector 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 5299)
  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 5299)
  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_051
FEATURES             Location/Qualifiers
     source          1..5299
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(307..1122)
                     /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(307..1122)
                     /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     1030..1049
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1114..1133
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1347..1935
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1836..1855
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_feature    2441..2942
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    3005..3038
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     RBS             3345..3356
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3363..4076
                     /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      4088..4159
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4175..4202
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    4575..4608
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    join(4780..5299,1..10)
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
ORIGIN
        1 ccctgctctg ggcttctggg tttgagtcct tggcaagccc aggagaggcg ctcaggcttc
       61 cctgtccccc ttcctcgtcc accatctcat gcccctggct ctcctgcccc ttccctacag
      121 gggttcctgg ctctgctcta cctactgcat taaaaaaaaa tttttttttt tctaagacaa
      181 ggtctgactc tgtctcccag gctggagtgc agtggtgcaa tcacgctcat agcctcaacc
      241 tcccgggctc cagtgggcct cccacctccg agcggccgcg tgttacaacc aattaaccaa
      301 ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
      361 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
      421 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
      481 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
      541 gacgactgaa tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac
      601 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
      661 tgattgcgcc tgagcgaggc gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
      721 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
      781 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
      841 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
      901 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
      961 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     1021 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     1081 tcgcggcctg gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     1141 gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta
     1201 acatcagaga ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt
     1261 gaaggatcag tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     1321 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     1381 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     1441 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     1501 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     1561 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     1621 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     1681 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     1741 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     1801 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     1861 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     1921 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     1981 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtgcggcc
     2041 gccaatccac aaccagtaag ggctggccca ggtgtctggc cccacagctg agctcctaac
     2101 cgcaccgagc ctctccaaaa catggtcttt ttttgctccc cactgccacc accatcaccg
     2161 tcccctgcct catccagtcc cccttcccat tctccagaga cttcctgggc catctccatg
     2221 accttccaga ccagcctggg cttcactctc aaatccaatg ctttctctga cctgcattct
     2281 ctcccctggg cctgtgccgc tttctgtctg cagcttgtgg cctgggtcac ctctacggct
     2341 ggcccagatc cttccctgcc gcctccttca ggttccgtct tcctccactc cctcttcccc
     2401 ttgctctctg ctgtgttgct gcccaaggat gctctttccg gagcacttcc ttctcggcgc
     2461 tgcaccacgt gatgtcctct gagcggatcc tccccgtgtc tgggtcctct ccgggcatct
     2521 ctcctccctc acccaacccc atgccgtctt cactcgctgg gttccctttt ccttctcctt
     2581 ctggggcctg tgccatctct cgtttcttag gatggccttc tccgacggat gtctcccttg
     2641 cgtcccgcct ccccttcttg taggcctgca tcatcaccgt ttttctggac aaccccaaag
     2701 taccccgtct gcctggcttt agccacctct ccatcctctt gctttctttg cctggacacc
     2761 ccgttctcct gtggattcgg gtcacctctc actcctttca tttgggcagc tcccctaccc
     2821 cccttacctc tctagtctgt gctagctctt ccagccccct gtcatggcat cttccagggg
     2881 tccgagagct cagctagtct tcttcctcca acccgggccc ctatgtccac ttcaggacag
     2941 catgtttgct gcctccaggg atcctgtgtc cccgagctgg gaccacctta tattcccagg
     3001 ccctgaagtt cctattctct agaaagtata ggaacttcct gatgagacgt ggtagagcca
     3061 caaacagccg gtacaagcaa cgatctccag gaccatctga atcatgcgcg gatgacacga
     3121 actcacgacg gcgatcacag acattaaccc acagtacaga cactgcgaca acgtggcaat
     3181 tcgtcgcaat acaacggaaa gtgaaacgtg atttcatgcg tcattttgaa cattttgtaa
     3241 atcttattta ataatgtgtg cggcaattca catttaattt atgaatgttt tcttaacatc
     3301 gcggcaactc aagaaacggc aggttcggat cttagctact agagaaagag gagaaatact
     3361 agatgcgtaa aggcgaagag ctgttcactg gtgtcgtccc tattctggtg gaactggatg
     3421 gtgatgtcaa cggtcataag ttttccgtgc gtggcgaggg tgaaggtgac gcaactaatg
     3481 gtaaactgac gctgaagttc atctgtacta ctggtaaact gccggttcct tggccgactc
     3541 tggtaacgac gctgacttat ggtgttcagt gctttgctcg ttatccggac catatgaagc
     3601 agcatgactt cttcaagtcc gccatgccgg aaggctatgt gcaggaacgc acgatttcct
     3661 ttaaggatga cggcacgtac aaaacgcgtg cggaagtgaa atttgaaggc gataccctgg
     3721 taaaccgcat tgagctgaaa ggcattgact ttaaagagga cggcaatatc ctgggccata
     3781 agctggaata caattttaac agccacaatg tttacatcac cgccgataaa caaaaaaatg
     3841 gcattaaagc gaattttaaa attcgccaca acgtggagga tggcagcgtg cagctggctg
     3901 atcactacca gcaaaacact ccaatcggtg atggtcctgt tctgctgcca gacaatcact
     3961 atctgagcac gcaaagcgtt ctgtctaaag atccgaacga gaaacgcgat catatggttc
     4021 tgctggagtt cgtaaccgca gcgggcatca cgcatggtat ggatgaactg tacaaatgac
     4081 caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     4141 tttgtcggtg aacgctctct actagagtca cactggctca ccttcgggtg ggcctttctg
     4201 cgtttatagc tggaaatctg ctcgtcagtg gtgctcacac tgacgaatca tgtacagatc
     4261 ataccgatga ctgcctggcg actcacaact aagcaagaca gccggaacca gcgccggcga
     4321 acaccactgc atatatggca tatcacaaca gtccacgtct caagcagtta cagagatgtt
     4381 acgaaccctg aacaggttga ctattaattg tgtctgcttg atgtggacac caggtggcgc
     4441 tggacatcag atttggagag gcagttgtct agggaaccgg gctctgtgcc agcgcaggag
     4501 gcaggctggc tctcctattc cagggatgct catccaggaa ggaaaggttg catgctggac
     4561 acactaacct tacagaagtt cctattctct agaaagtata ggaacttcga gtgattggtg
     4621 acagaaaagc cccatcctta ggcctcctcc ttcctagtct cctgatattg ggtctaaccc
     4681 ccacctcctg ttaggcagat tccttatctg gtgacacacc cccatttcct ggagccatct
     4741 ctctccttgc cagaacctct aaggtttgct tacgatggag ccagagagga tcctgggagg
     4801 gagagcttgg cagggggtgg gagggaaggg ggggatgcgt gacctgcccg gttctcagtg
     4861 gccaccctgc gctaccctct cccagaacct gagctgctct gacgcggccg tctggtgcgt
     4921 ttcactgatc ctggtgctgc agcttcctta cacttcccaa gaggagaagc agtttggaaa
     4981 aacaaaatca gaataagttg gtcctgagtt ctaactttgg ctcttcacct ttctagtccc
     5041 caatttatat tgttcctccg tgcgtcagtt ttacctgtga gataaggcca gtagccagcc
     5101 ccgtcctggc agggctgtgg tgaggagggg ggtgtccgtg tggaaaactc cctttgtgag
     5161 aatggtgcgt cctaggtgtt caccaggtcg tggccgcctc tactcccttt ctctttctcc
     5221 atccttcttt ccttaaagag tccccagtgc tatctgggac atattcctcc gcccagagca
     5281 gggtcccgct tccctaagg
//
