LOCUS       sequence-236791-        6694 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the phiC31-hygromycin-mRuby2PEST 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 6694)
  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 6694)
  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_030
FEATURES             Location/Qualifiers
     source          1..6694
                     /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)."
     enhancer        2708..3011
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3013..3288
                     /label=chicken beta-actin promoter
     intron          3289..4301
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4225..4247
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4309..4328
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             4413..5432
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4413..5432
                     /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             5448..5501
                     /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             5508..5528
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5544..6251
                     /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(6412..6431)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6466..6485
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
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 ccatatatgg
     2701 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
     2761 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
     2821 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
     2881 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
     2941 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
     3001 ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct
     3061 ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg
     3121 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg
     3181 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
     3241 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
     3301 ttgccttcgc cccgtgcccc gctccgcgcc gcctcgcgcc gcccgccccg gctctgactg
     3361 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     3421 cgcttggttt aatgacggct cgtttctttt ctgtggctgc gtgaaagcct taaagggctc
     3481 cgggagggcc ctttgtgcgg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt
     3541 ggggagcgcc gcgtgcggcc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
     3601 gggctttgtg cgctccgcgt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt
     3661 gcgggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc
     3721 agggggtgtg ggcgcggcgg tcgggctgta acccccccct gcacccccct ccccgagttg
     3781 ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg
     3841 tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg
     3901 gggagggctc gggggagggg cgcggcggcc cccggagtgc cggcggctgt cgaggcgcgg
     3961 cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt
     4021 cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg
     4081 gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc
     4141 cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc
     4201 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag
     4261 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     4321 ttattgtgct gtctcatcat tttggcaaag aattcgccac tatggcccca actggggtaa
     4381 cctttgagtt ctctcagttg gggggttctg gtaaaaagcc tgaactcacc gcgacgtctg
     4441 tcgagaagtt tctgatcgaa aagttcgaca gcgtgtccga cctgatgcag ctctcggagg
     4501 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     4561 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     4621 cgctcccgat tccggaagtg cttgacattg gggaatttag cgagagcctg acctattgca
     4681 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     4741 ttctgcaacc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     4801 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     4861 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     4921 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     4981 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     5041 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     5101 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     5161 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     5221 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     5281 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     5341 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     5401 gacgccccag cactcgtccg agggcaaagg aaggatccgg aagcggagag ggcagaggaa
     5461 gtctgctaac atgcggtgac gtcgaggaga atcctggacc tggatccccc aagaagaaga
     5521 ggaaggtgga ggacgccgga tccgtgtcca aaggagagga gttaatcaag gaaaacatga
     5581 gaatgaaagt tgtcatggag ggctccgtta atggtcacca attcaagtgt acaggggaag
     5641 gtgaaggtaa tccttacatg ggtacacaaa ctatgagaat taaagtaatt gaaggcggac
     5701 cactaccatt tgcatttgac attctggcaa cgtcattcat gtacggatca cgaactttca
     5761 tcaagtaccc taaaggtata ccagactttt tcaagcaatc ttttccagag ggttttacat
     5821 gggaaagggt tacaagatac gaagatgggg gtgtcgtcac agttatgcaa gatacttcat
     5881 tagaagatgg ctgccttgtc tatcatgtgc aagtaagagg ggtgaatttt ccttctaacg
     5941 gacctgtgat gcagaaaaag accaaaggtt gggaaccaaa tactgaaatg atgtacccag
     6001 ctgatggagg tttgagaggc tacacacaca tggcgcttaa agttgatggt ggaggtcatt
     6061 tgtcttgtag ttttgttacc acttatcgtt ctaaaaagac tgttggcaat atcaaaatgc
     6121 caggaataca tgctgtagac cacagactag aaagactcga agagagcgat aacgaaatgt
     6181 tcgttgtaca gagagagcat gccgtagcca aatttgctgg cttaggcggt ggtatggatg
     6241 aattgtataa gggatcccat ggcttcccgc cggaggtgga ggagcaggat gatggcgcgc
     6301 tgcccatgtc ttgtgcccag gagagcggga tggaccgtca ccctgcagcc tgtgcttctg
     6361 ctaggatcaa tgtgtaactc gagtggcgat ccagacatga taagatacat tgatgagttt
     6421 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     6481 attgctttat ttgtaaccat tataagctgc aataaacgct gagcatgaga cggaaatctg
     6541 ctcgtcagtg gtgctcacac tgacgaatca tgtacagatc ataccgatga ctgcctggcg
     6601 actcacaact aagcaagaca gccggaacca gcgccggcga acaccactgc atatatggca
     6661 tatcacaaca gtccaactag tgcactgcag taca
//
