LOCUS       sequence-261577-        8442 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  TET3G-2xMS2(wt+f6), NLS-MCP-ANR-ANR-P2a-BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8442)
  AUTHORS   Foight GW, Wang Z, Wei CT, Jr Greisen P, Warner KM, 
            Cunningham-Bryant D, Park K, Brunette TJ, Sheffler W, Baker D, Maly 
            DJ
  TITLE     Multi-input chemical control of protein dimerization for programming
            graded cellular responses.
  JOURNAL   Nat Biotechnol. 2019 Sep 9. pii: 10.1038/s41587-019-0242-8. doi: 
            10.1038/s41587-019-0242-8.
  PUBMED    31501561
REFERENCE   2  (bases 1 to 8442)
  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"; journalName: "Nat 
            Biotechnol. 2019 Sep 9. pii: 10.1038/s41587-019-0242-8. doi: 
            10.1038/s41587-019-0242-8."
COMMENT     Sequence Label: GWF297
FEATURES             Location/Qualifiers
     source          1..8442
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..200
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             218..398
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    445..570
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1067..1300
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1485..1529
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1827..1944
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2197..2230)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2234..2254
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        2430..2448
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     terminator      2480..2499
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     enhancer        2631..2934
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2935..3138
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3175..3195
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3847..3903
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3904..4602
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(4625..4658)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4714..5302
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4767..4787)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5185..5196)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5305..5321)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5306..5322)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5831..6011
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6073..6661)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6153..6172)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6832..7623,7624..7692))
                     /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     7455..7474
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7693..7797)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7872..7891)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        8063..8442
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag cgcgttttgc ctgtactggg tctctctggt tagaccagat
      241 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
      301 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
      361 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg
      421 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc
      481 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc
      541 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga
      601 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta
      661 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa
      721 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt
      781 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa
      841 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca
      901 cagcaagcgg ccggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
      961 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     1021 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     1081 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     1141 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     1201 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     1261 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     1321 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     1381 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     1441 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     1501 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     1561 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     1621 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     1681 acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa ggtggagaga
     1741 gagacagaga cagatccatt cgattagtga acggatcggc actgcgtgcg ccaattctgc
     1801 agacaaatgg cagtattcat ccacaatttt aaaagaaaag gggggattgg ggggtacagt
     1861 gcaggggaaa gaatagtaga cataatagca acagacatac aaactaaaga attacaaaaa
     1921 caaattacaa aaattcaaaa ttttcgggtt tattacaggg acagcagaga tccagtttgg
     1981 ttagtaccgg gcccgctcta gagatccgac gcgccatctc taggcccgcg ccggccccct
     2041 cgcacggact tgtgggagaa gctcggctac tcccctgccc cggttaattt gcatataata
     2101 tttcctagta actatagagg cttaatgtgc gataaaagac agataatctg ttctttttaa
     2161 tactagctac attttacatg ataggcttgg atttctataa cttcgtatag catacattat
     2221 acgaagttat aaacagcaca aaaggaaact caccctaact gtaaagtaat tgtgtgtttt
     2281 gagactataa gtatcccttg gagaaccacc ttgttggtac gttctctatc actgatagtt
     2341 taagagctat gctggaaaca gcatagcaag tttaaataag gctagtccgt tatcaacttg
     2401 aaaaagtggc accgagtcgg tgcgggagca catgaggatc acccatgtgc gactcccaca
     2461 gtcactgggg agtcttccct ttttttgttt tttatgtctc tgcagagttc ggtctcgagt
     2521 actaggatcc attaggcggc cgcgtggata accgtattac cgccatgcat tagttattaa
     2581 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     2641 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     2701 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     2761 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     2821 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     2881 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     2941 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     3001 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     3061 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     3121 gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcccac catgcccaaa
     3181 aagaaaagaa aagtgggtag tatggcttct aactttactc agttcgttct cgtcgacaat
     3241 ggcggaactg gcgacgtgac tgtcgcccca agcaacttcg ctaacgggat cgctgaatgg
     3301 atcagctcta actcgcgttc acaggcttac aaagtaacct gtagcgttcg tcagagctct
     3361 gcgcagaatc gcaaatacac catcaaagtc gaggtgccta aaggcgcctg gcgttcgtac
     3421 ttaaatatgg aactaaccat tccaattttc gccacgaatt ccgactgcga gcttattgtt
     3481 aaggcaatgc aaggtctcct aaaagatgga aacccgattc cctcagcaat cgcagcaaac
     3541 tccggcatct acggctccgg aggttccggg acgggatctg gtactggttc tggtaccggg
     3601 acaacgtccg gaacagggac agggggctcc actgggggtg aattggacga gttggtttat
     3661 ctgctcgacg ggcctggtta tgatcccatc cattccgatg gaagcggtac tggtagcggc
     3721 acaggctccg gaacgggcac gacgagcggt acaggaacag gtggtagtac aggtggggag
     3781 cttgatgagc ttgtctacct ccttgacggc cccggttacg accctatcca tagcgacgga
     3841 agcggagcta ctaacttcag cctgctgaag caggctggag acgtggagga gaaccctgga
     3901 cctagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac
     3961 aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
     4021 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact
     4081 agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc
     4141 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
     4201 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
     4261 atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg
     4321 gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg
     4381 gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac atatagatcc
     4441 aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa
     4501 agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga
     4561 tactgcgacc tccctagcaa actggggcac aagcttaatt gataagaatt cgtcgaggga
     4621 cctaataact tcgtatagca tacattatac gaagttatac atgtttaagg gttccggttc
     4681 cactaggtac aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt
     4741 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     4801 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     4861 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     4921 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     4981 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     5041 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     5101 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     5161 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     5221 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     5281 tccctttggg ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc tagaaaaaca
     5341 tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc
     5401 acaagaggag gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac
     5461 ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct
     5521 aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta
     5581 cttccctgat tggcagaact acacaccagg gccagggatc agatatccac tgacctttgg
     5641 atggtgctac aagctagtac cagttgagca agagaaggta gaagaagcca atgaaggaga
     5701 gaacacccgc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt
     5761 attagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag agctgcatcc
     5821 ggactgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     5881 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     5941 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     6001 aatctctagc agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     6061 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6121 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6181 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6241 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6301 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6361 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6421 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6481 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6541 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6601 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6661 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6721 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6781 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6841 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6901 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6961 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     7021 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     7081 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     7141 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7201 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7261 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7321 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7381 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7441 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7501 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7561 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7621 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7681 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7741 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7801 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     7861 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     7921 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc
     7981 ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat
     8041 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     8101 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     8161 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     8221 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     8281 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     8341 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     8401 acttggcagt acatctacgt attagtcatc gctattacca tg
//
