LOCUS       sequence-251663-        5542 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes BxBI landing pad 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 5542)
  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 5542)
  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_010
FEATURES             Location/Qualifiers
     source          1..5542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(103..894,895..963))
                     /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     726..745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(964..1068)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1171..1759
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1660..1679
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2653..2686
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2703..3213
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3132..3150
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     protein_bind    3220..3267
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             3278..4297
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3278..4297
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4313..4366
                     /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             4373..4393
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4409..5116
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTHM
                     ALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAVA
                     KFAGLGGGMDELYK"
     primer_bind     complement(5135..5152)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5141..5365
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gcggccgcga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
       61 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
      121 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
      181 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
      241 gcgggaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
      301 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
      361 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
      421 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
      481 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
      541 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
      601 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
      661 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
      721 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
      781 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
      841 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
      901 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
      961 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     1021 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     1081 aaaagtgcca cctgtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1141 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1201 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1261 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     1321 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     1381 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1441 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     1501 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     1561 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     1621 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     1681 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     1741 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     1801 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtgc
     1861 ggccgcccct gaattcgcat ctagatggta gagccacaaa cagccggtac aagcaacgat
     1921 ctccaggacc atctgaatca tgcgcggatg acacgaactc acgacggcga tcacagacat
     1981 taacccacag tacagacact gcgacaacgt ggcaattcgt cgcaataccg tctcactgaa
     2041 ctggccgata attgcagacg atcgaattcg agttggcgcg cctgtcattc taaatctctc
     2101 tttcagccta aagctttttc cccgtatccc cccaggtgtc tgcaggctca aagagcagcg
     2161 agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc tgtccccgca
     2221 cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc gggcggctcg
     2281 ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg ggagggggct
     2341 gtccccgtga gctcccaggc gcgcctgtca ttctaaatct ctctttcagc ctaaagcttt
     2401 ttccccgtat ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa
     2461 agcgatcccg tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga
     2521 tgcgggggga gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg
     2581 gggagggacg taattacatc cctgggggct ttgggggggg gctgtccccg tgagctcaat
     2641 aaaagagccc acataacttc gtataatgta tgctatacga agttataacg tcgaattccc
     2701 acggggttgg ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc
     2761 tctgggcgtg gttccgggaa acgcagcggc gccgaccctg ggtcttgcac attcttcacg
     2821 tccgttcgca gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt
     2881 cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa
     2941 acggaagccg cacgtgtcac tagtaccctc gcagacggac agcgccaggg agcaatggca
     3001 gcgcgccgac cgcgatgggc tgtggccaat agcggctgct cagcagggcg cgccgagagc
     3061 agcggccggg aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc
     3121 ctgcccgcgc ggtgttccgc attctgcaag cctccggagc gcacgtcggc agtcggctcc
     3181 ctcgttgacc gaatcaccga cctctctccc cagtatggtg gtttgtctgg tcaaccaccg
     3241 cggtctcagt ggtgtacggt acaaaccagg ttctggtaaa aagcctgaac tcaccgcgac
     3301 gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtg tccgacctga tgcagctctc
     3361 ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg
     3421 ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc
     3481 ggccgcgctc ccgattccgg aagtgcttga cattggggaa tttagcgaga gcctgaccta
     3541 ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc
     3601 cgctgttctg caaccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca
     3661 gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga
     3721 tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac
     3781 cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc
     3841 cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg
     3901 ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt
     3961 cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt
     4021 cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat
     4081 tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc
     4141 gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat
     4201 cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg
     4261 aaaccgacgc cccagcactc gtccgagggc aaaggaagga tccggaagcg gagagggcag
     4321 aggaagtctg ctaacatgcg gtgacgtcga ggagaatcct ggacctggat cccccaagaa
     4381 gaagaggaag gtggaggacg ccggatccgt gtccaaagga gaggagttaa tcaaggaaaa
     4441 catgagaatg aaagttgtca tggagggctc cgttaatggt caccaattca agtgtacagg
     4501 ggaaggtgaa ggtaatcctt acatgggtac acaaactatg agaattaaag taattgaagg
     4561 cggaccacta ccatttgcat ttgacattct ggcaacgtca ttcatgtacg gatcacgaac
     4621 tttcatcaag taccctaaag gtataccaga ctttttcaag caatcttttc cagagggttt
     4681 tacatgggaa agggttacaa gatacgaaga tgggggtgtc gtcacagtta tgcaagatac
     4741 ttcattagaa gatggctgcc ttgtctatca tgtgcaagta agaggggtga attttccttc
     4801 taacggacct gtgatgcaga aaaagaccaa aggttgggaa ccaaatactg aaatgatgta
     4861 cccagctgat ggaggtttga gaggctacac acacatggcg cttaaagttg atggtggagg
     4921 tcatttgtct tgtagttttg ttaccactta tcgttctaaa aagactgttg gcaatatcaa
     4981 aatgccagga atacatgctg tagaccacag actagaaaga ctcgaagaga gcgataacga
     5041 aatgttcgtt gtacagagag agcatgccgt agccaaattt gctggcttag gcggtggtat
     5101 ggatgaattg tataagtaac tcgagtggcc gacgcctcga ctgtgccttc tagttgccag
     5161 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     5221 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     5281 ctggggggtg gggtggtgca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     5341 gctggggatg cggtgggctc tatgggctga gcatgagacg gaaatctgct cgtcagtggt
     5401 gctcacactg acgaatcatg tacagatcat accgatgact gcctggcgac tcacaactaa
     5461 gcaagacagc cggaaccagc gccggcgaac accactgcat atatggcata tcacaacagt
     5521 ccaactagtg cactgcagta ca
//
