LOCUS       sequence-474932-        7270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-overexpression of TOMM20-Rhobin9 fusion protein and 
            NLS-mTagBFP-2xNLS as a nuclear labeling marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7270)
  AUTHORS   Chen Y, Yserentant K, Hong K, Kuang Y, Bhowmick A, Charles-Orszag A,
            Lord SJ, Lu L, Hou K, Mann SI, Grimm JB, Lavis LD, Mullins RD, 
            DeGrado WF, Huang B
  TITLE     De novo designed bright, hyperstable rhodamine binders for 
            fluorescence microscopy
  JOURNAL   bioRxiv 2025.06.24.661379
REFERENCE   2  (bases 1 to 7270)
  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: "bioRxiv 
            2025.06.24.661379"
COMMENT     Sequence Label: pTOMM20-Rhobin9-IRES-NLS-mTagBFP-2xNLS
FEATURES             Location/Qualifiers
     source          1..7270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(746..814,815..1606)
                     /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     complement(964..983)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      1619..1755
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1907..1978
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(1983..2029)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    2047..2095
                     /note="synthetic polyadenylation signal"
     enhancer        2176..2479
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2481..2683
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2634..2654
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    3677..4250
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3700..4252
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3844..3861)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4071..4090
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4270..4290
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4315..5013
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             5023..5070
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5080..5100
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5153..5274
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5164..5183)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5218..5237
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      5389..5435
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      5465..5601
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5646..5717
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(6682..7270)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6762..6781)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcattt
      241 aaatttccga actctccaag gccctcgtcg gaaaatcttc aaacctttcg tccgatccat
      301 cttgcaggct acctctcgaa cgaactatcg caagtctctt ggccggcctt gcgccttggc
      361 tattgcttgg cagcgcctat cgccaggtat tactccaatc ccgaatatcc gagatcggga
      421 tcacccgaga gaagttcaac ctacatcctc aatcccgatc tatccgagat ccgaggaata
      481 tcgaaatcgg ggcgcgcctg gtgtaccgag aacgatcctc tcagtgcgag tctcgacgat
      541 ccatatcgtt gcttggcagt cagccagtcg gaatccagct tgggacccag gaagtccaat
      601 cgtcagatat tgtactcaag cctggtcacg gcagcgtacc gatctgttta aacctagata
      661 ttgatagtct gatcggtcaa cgtataatcg agtcctagct tttgcaaaca tctatcaaga
      721 gacaggatca gcaggaggct ttcgcatgag tattcaacat ttccgtgtcg cccttattcc
      781 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
      841 agatgctgaa gatcagttgg gtgcgcgagt gggttacatc gaactggatc tcaacagcgg
      901 taagatcctt gagagttttc gccccgaaga acgctttcca atgatgagca cttttaaagt
      961 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     1021 catacactat tctcagaatg acttggttga gtattcacca gtcacagaaa agcatcttac
     1081 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     1141 ggccaactta cttctgacaa cgattggagg accgaaggag ctaaccgctt ttttgcacaa
     1201 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     1261 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaaccttgc gtaaactatt
     1321 aactggcgaa ctacttactc tagcttcccg gcaacagttg atagactgga tggaggcgga
     1381 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     1441 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     1501 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     1561 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaaccga ttctaggtgc
     1621 attggcgcag aaaaaaatgc ctgatgcgac gctgcgcgtc ttatactccc acatatgcca
     1681 gattcagcaa cggatacggc ttccccaact tgcccacttc catacgtgtc ctccttacca
     1741 gaaatttatc cttaagatcc cgaatcgttt aaactcgact ctggctctat cgaatctccg
     1801 tcgtttcgag cttacgcgaa cagccgtggc gctcatttgc tcgtcgggca tcgaatctcg
     1861 tcagctatcg tcagcttacc tttttggcag cgatcgcggc tcccgacatc ttggaccatt
     1921 agctccacag gtatcttctt ccctctagtg gtcataacag cagcttcagc tacctctcaa
     1981 ttcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctat caatcgttgc
     2041 gttacacaca caaaaaacca acacacatcc atcttcgatg gatagcgatt ttattatcta
     2101 actgctgatc gagtgtagcc agatctagta atcaattacg gggtcattag ttcatagccc
     2161 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     2221 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     2281 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     2341 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     2401 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     2461 agtcatcgct attaccatgc tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     2521 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     2581 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     2641 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
     2701 gatcagatct ttgtcgatcc taccatccac tcgacacacc cgccaggcgg gggtggctca
     2761 ggtggcatgg tgggtcggaa cagcgccatc gccgccggtg tatgcggggc ccttttcatt
     2821 gggtactgca tctacttcga ccgcaaaaga cgaagtgacc ccaacttcaa gaacaggctt
     2881 cgagaacgaa gaaagaaaca gaagcttgcc aaggagagag ctgggctttc caagttacct
     2941 gaccttaaag atgctgaagc tgttcagaag ttcttccttg aagaaataca gcttggtgaa
     3001 gagttactag ctcaaggtga atatgagaag ggcgtagacc atctgacaaa tgcaattgct
     3061 gtgtgtggac agccacagca gttactgcag gtcttacagc aaactcttcc accaccagtg
     3121 ttccagatgc ttctgactaa gctcccaaca attagtcaga gaattgtaag tgctcagagc
     3181 ttggctgaag atgatgtgga aggcgggggg tctgggggag ggggatccgg gatggaaaaa
     3241 aaaaagaagc ttatcgagat ctatgagaaa attattggat acctcaagaa gctgtatgac
     3301 cttgcaagga aactcgcgga agcccttagg gcaggcgaca gggaaagggc tgggaagctg
     3361 ttggatgaat ttcttgctgt atacaatgaa atggtcgcca ttgggaaaaa aatgttggag
     3421 gaggccaaaa aggtattcac agaggaggaa atacagaaga tatttgaaga gaacgatgta
     3481 aaaaggaagg aattgggcgt agatttgaat gcagcggagg ttgatcggct ggttgcggag
     3541 cttcgagccg cgttcgaggc cggagatacg gcgagggcag cagaaatcgt tgaacggttg
     3601 gttaaattct accaggcgaa cattatcatc ggtgaggagc ggatcaagaa gctcaaatct
     3661 gaattgtgag gatccgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct
     3721 tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg
     3781 gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt
     3841 cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg
     3901 aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac
     3961 ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg
     4021 cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct
     4081 caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg
     4141 atctggggcc tcggtacaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag
     4201 gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccgag
     4261 ctcatggccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccatgagc
     4321 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
     4381 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     4441 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     4501 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     4561 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     4621 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     4681 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     4741 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     4801 aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag atccaagaaa
     4861 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     4921 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     4981 gacctcccta gcaaactggg gcacaagctt aatggtggag gtaaaaggcc ggcggccacg
     5041 aaaaaggccg gccaggcaaa aaagaaaaag ggtggaggtc caaaaaagaa aaggaaggtc
     5101 taagctagct tgactgactg agatacagcg taccttcagc tcacagacat gataagatac
     5161 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     5221 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     5281 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     5341 aagtaaaacc tctacaaatg tggtattggc ccatctctat cggtatcgta gcataacccc
     5401 ttggggcctc taaacgggtc ttgaggggtt ttttgtgccc ctcgggccgg attgctatct
     5461 accggcattg gcgcagaaaa aaatgcctga tgcgacgctg cgcgtcttat actcccacat
     5521 atgccagatt cagcaacgga tacggcttcc ccaacttgcc cacttccata cgtgtcctcc
     5581 ttaccagaaa tttatcctta aggtcgtcag ctatcctgca ggcgatctct cgatttcgat
     5641 caagacattc ctttaatggt cttttctgga caccactagg ggtcagaagt agttcatcaa
     5701 actttcttcc ctccctaatc tcattggtta ccttgggcta tcgaaactta attaagtagg
     5761 tgggcgggcc aagatagggg cgcgattgct gcgatctgga ggacaaatta cacacacttg
     5821 cgcctgagcg ccaagcacag ggttgttggt cctcatattc acgaggtcgc tgagagcacg
     5881 gtgggctaat gttgccatgg gtagcatata ctacccaaat atctggatag catatgctat
     5941 cctaatctat atctgggtag cataggctat cctaatctat atctgggtag catatgctat
     6001 cctaatctat atctgggtag tatatgctat cctaatttat atctgggtag cataggctat
     6061 cctaatctat atctgggtag catatgctat cctaatctat atctgggtag tatatgctat
     6121 cctaatctgt atccgggtag catatgctat cctaatagag attagggtag tatatgctat
     6181 cctaatttat atctgggtag catatactac ccaaatatct ggatagcata tgctatccta
     6241 atctatatct gggtagcata tgctatccta atctatatct gggtagcata ggctatccta
     6301 atctatatct gggtagcata tgctatccta atctatatct gggtagtata tgctatccta
     6361 atttatatct gggtagcata ggctatccta atctatatct gggtagcata tgctatccta
     6421 atctatatct gggtagtata tgctatccta atctgtatcc gggtagcata tgctatcctc
     6481 atgatattaa ttaaccagtc aagtcagcta cttggcgaga tcgacttgtc tgggtttcga
     6541 ctacgctcag aattgcgtca gtcaagttcg atctggtcct tgctattgca cccgttctcc
     6601 gattacgagt ttcatttaaa tcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6661 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6721 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6781 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6841 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6901 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6961 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     7021 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     7081 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     7141 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     7201 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     7261 aggatctcaa
//
