LOCUS       sequence-320322-        6711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes the BxBI-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 6711)
  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 6711)
  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_031
FEATURES             Location/Qualifiers
     source          1..6711
                     /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..3289
                     /label=chicken beta-actin promoter
     intron          3290..4306
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4230..4252
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4314..4333
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    4372..4419
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             4430..5449
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4430..5449
                     /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             5465..5518
                     /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             5525..5545
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5561..6268
                     /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(6429..6448)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6483..6502
                     /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 ycgggctgaa 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 gggsrcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag
     3181 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
     3241 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
     3301 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
     3361 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
     3421 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
     3481 gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg
     3541 cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg
     3601 cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc
     3661 gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg
     3721 gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg
     3781 agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct
     3841 cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg
     3901 ggccggggag ggctcggggg aggggcgcgg cggcccccgg agtgccggcg gctgtcgagg
     3961 cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc
     4021 tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg
     4081 gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc
     4141 gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg
     4201 ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc
     4261 tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt
     4321 gctggttatt gtgctgtctc atcattttgg caaagaattc gccactatgg tggtttgtct
     4381 ggtcaaccac cgcggtctca gtggtgtacg gtacaaacca ggttctggta aaaagcctga
     4441 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tgtccgacct
     4501 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg
     4561 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg
     4621 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aatttagcga
     4681 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga
     4741 aaccgaactg cccgctgttc tgcaaccggt cgcggaggcc atggatgcga tcgctgcggc
     4801 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac
     4861 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt
     4921 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
     4981 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct
     5041 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc
     5101 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
     5161 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
     5221 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
     5281 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
     5341 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
     5401 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaag gatccggaag
     5461 cggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggacctgg
     5521 atcccccaag aagaagagga aggtggagga cgccggatcc gtgtccaaag gagaggagtt
     5581 aatcaaggaa aacatgagaa tgaaagttgt catggagggc tccgttaatg gtcaccaatt
     5641 caagtgtaca ggggaaggtg aaggtaatcc ttacatgggt acacaaacta tgagaattaa
     5701 agtaattgaa ggcggaccac taccatttgc atttgacatt ctggcaacgt cattcatgta
     5761 cggatcacga actttcatca agtaccctaa aggtatacca gactttttca agcaatcttt
     5821 tccagagggt tttacatggg aaagggttac aagatacgaa gatgggggtg tcgtcacagt
     5881 tatgcaagat acttcattag aagatggctg ccttgtctat catgtgcaag taagaggggt
     5941 gaattttcct tctaacggac ctgtgatgca gaaaaagacc aaaggttggg aaccaaatac
     6001 tgaaatgatg tacccagctg atggaggttt gagaggctac acacacatgg cgcttaaagt
     6061 tgatggtgga ggtcatttgt cttgtagttt tgttaccact tatcgttcta aaaagactgt
     6121 tggcaatatc aaaatgccag gaatacatgc tgtagaccac agactagaaa gactcgaaga
     6181 gagcgataac gaaatgttcg ttgtacagag agagcatgcc gtagccaaat ttgctggctt
     6241 aggcggtggt atggatgaat tgtataaggg atcccatggc ttcccgccgg aggtggagga
     6301 gcaggatgat ggcgcgctgc ccatgtcttg tgcccaggag agcgggatgg accgtcaccc
     6361 tgcagcctgt gcttctgcta ggatcaatgt gtaactcgag tggcgatcca gacatgataa
     6421 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     6481 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacgctgag
     6541 catgagacgg aaatctgctc gtcagtggtg ctcacactga cgaatcatgt acagatcata
     6601 ccgatgactg cctggcgact cacaactaag caagacagcc ggaaccagcg ccggcgaaca
     6661 ccactgcata tatggcatat cacaacagtc caactagtgc actgcagtac a
//
