LOCUS       sequence-474930-        7269 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-overexpression of TOMM20-Rhobin2 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 7269)
  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 7269)
  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-Rhobin2-IRES-NLS-mTagBFP-2xNLS
FEATURES             Location/Qualifiers
     source          1..7269
                     /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    3676..4249
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3699..4251
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3843..3860)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4070..4089
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4269..4289
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4314..5012
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             5022..5069
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5079..5099
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5152..5273
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5163..5182)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5217..5236
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      5388..5434
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      5464..5600
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5645..5716
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(6681..7269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6761..6780)
                     /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 ggtggctcag
     2761 gtggcatggt gggtcggaac agcgccatcg ccgccggtgt atgcggggcc cttttcattg
     2821 ggtactgcat ctacttcgac cgcaaaagac gaagtgaccc caacttcaag aacaggcttc
     2881 gagaacgaag aaagaaacag aagcttgcca aggagagagc tgggctttcc aagttacctg
     2941 accttaaaga tgctgaagct gttcagaagt tcttccttga agaaatacag cttggtgaag
     3001 agttactagc tcaaggtgaa tatgagaagg gcgtagacca tctgacaaat gcaattgctg
     3061 tgtgtggaca gccacagcag ttactgcagg tcttacagca aactcttcca ccaccagtgt
     3121 tccagatgct tctgactaag ctcccaacaa ttagtcagag aattgtaagt gctcagagct
     3181 tggctgaaga tgatgtggaa ggcggggggt ctgggggagg gggatccggg atggagaaaa
     3241 atcagtttat catcgatatt tatgaaaagt tgaatgagta ccacaagaag ctgctcgaac
     3301 tggcgagagc gctggcagaa gcacttcggg ccggggatcg agcgcgcgct ggccgattgc
     3361 tggaggagtt ccttgcgacg ttcaaagcaa tgaaagcata cggagatgct aaagaggtcg
     3421 agagttttct ctacttcagt gaggaggaac ttgcggccat atttgcggaa tcagatgtcg
     3481 aacggaagaa gctgggagtg acactcaaga tccctgaagt cgtggccatg gtagaggaac
     3541 tgggtcgcgc atttgaggct ggggatactg ctactgcggc ggcgatagta gaaaagttgg
     3601 taaagtttta cgaggcaaac ataataatag gccaaaagag aatcgatgaa ctcaagtcaa
     3661 aaatttgagg atccgcccct ctccctcccc cccccctaac gttactggcc gaagccgctt
     3721 ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg
     3781 caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc
     3841 ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga
     3901 agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc
     3961 tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc
     4021 acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc
     4081 aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga
     4141 tctggggcct cggtacacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg
     4201 ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccgagc
     4261 tcatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccatgagcg
     4321 agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc
     4381 acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa
     4441 tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc
     4501 tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt
     4561 ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga
     4621 ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag
     4681 gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct
     4741 tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga
     4801 agctcgtggg cgggagccat ctgatcgcaa acatcaagac cacatataga tccaagaaac
     4861 ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca
     4921 aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg
     4981 acctccctag caaactgggg cacaagctta atggtggagg taaaaggccg gcggccacga
     5041 aaaaggccgg ccaggcaaaa aagaaaaagg gtggaggtcc aaaaaagaaa aggaaggtct
     5101 aagctagctt gactgactga gatacagcgt accttcagct cacagacatg ataagataca
     5161 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     5221 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     5281 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     5341 agtaaaacct ctacaaatgt ggtattggcc catctctatc ggtatcgtag cataacccct
     5401 tggggcctct aaacgggtct tgaggggttt tttgtgcccc tcgggccgga ttgctatcta
     5461 ccggcattgg cgcagaaaaa aatgcctgat gcgacgctgc gcgtcttata ctcccacata
     5521 tgccagattc agcaacggat acggcttccc caacttgccc acttccatac gtgtcctcct
     5581 taccagaaat ttatccttaa ggtcgtcagc tatcctgcag gcgatctctc gatttcgatc
     5641 aagacattcc tttaatggtc ttttctggac accactaggg gtcagaagta gttcatcaaa
     5701 ctttcttccc tccctaatct cattggttac cttgggctat cgaaacttaa ttaagtaggt
     5761 gggcgggcca agataggggc gcgattgctg cgatctggag gacaaattac acacacttgc
     5821 gcctgagcgc caagcacagg gttgttggtc ctcatattca cgaggtcgct gagagcacgg
     5881 tgggctaatg ttgccatggg tagcatatac tacccaaata tctggatagc atatgctatc
     5941 ctaatctata tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc
     6001 ctaatctata tctgggtagt atatgctatc ctaatttata tctgggtagc ataggctatc
     6061 ctaatctata tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc
     6121 ctaatctgta tccgggtagc atatgctatc ctaatagaga ttagggtagt atatgctatc
     6181 ctaatttata tctgggtagc atatactacc caaatatctg gatagcatat gctatcctaa
     6241 tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagcatag gctatcctaa
     6301 tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagtatat gctatcctaa
     6361 tttatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa
     6421 tctatatctg ggtagtatat gctatcctaa tctgtatccg ggtagcatat gctatcctca
     6481 tgatattaat taaccagtca agtcagctac ttggcgagat cgacttgtct gggtttcgac
     6541 tacgctcaga attgcgtcag tcaagttcga tctggtcctt gctattgcac ccgttctccg
     6601 attacgagtt tcatttaaat catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     6661 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6721 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6781 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6841 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6901 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6961 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     7021 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     7081 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     7141 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     7201 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     7261 ggatctcaa
//
