LOCUS       sequence-474935-        7924 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-overexpression of Rhobin2-H2B-mNeonGreen3K 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 7924)
  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 7924)
  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: pRhobin2-H2B-mNG3K-IRES-NLS-mTagBFP-2xNLS
FEATURES             Location/Qualifiers
     source          1..7924
                     /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"
     CDS             3229..3603
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     regulatory      3889..3898
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    4331..4904
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    4354..4906
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4498..4515)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4725..4744
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4924..4944
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4969..5667
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             5677..5724
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5734..5754
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5807..5928
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5818..5837)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5872..5891
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      6043..6089
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      6119..6255
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    6300..6371
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(7336..7924)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7416..7435)
                     /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 cgccagcggc cgctgccaag
     2761 cttatggaga aaaatcagtt tatcatcgat atttatgaaa agttgaatga gtaccacaag
     2821 aagctgctcg aactggcgag agcgctggca gaagcacttc gggccgggga tcgagcgcgc
     2881 gctggccgat tgctggagga gttccttgcg acgttcaaag caatgaaagc atacggagat
     2941 gctaaagagg tcgagagttt tctctacttc agtgaggagg aacttgcggc catatttgcg
     3001 gaatcagatg tcgaacggaa gaagctggga gtgacactca agatccctga agtcgtggcc
     3061 atggtagagg aactgggtcg cgcatttgag gctggggata ctgctactgc ggcggcgata
     3121 gtagaaaagt tggtaaagtt ttacgaggca aacataataa taggccaaaa gagaatcgat
     3181 gaactcaagt caaaaattgg cggtaccggc gggggtggct caggtggccc agagccagcg
     3241 aagtctgctc ccgccccgaa aaagggctcc aagaaggcgg tgactaaggc gcagaagaaa
     3301 ggcggcaaga agcgcaagcg cagccgcaag gagagctatt ccatctatgt gtacaaggtt
     3361 ctgaagcagg tccaccctga caccggcatt tcgtccaagg ccatgggcat catgaattcg
     3421 tttgtgaacg acattttcga gcgcatcgca ggtgaggctt cccgcctggc gcattacaac
     3481 aagcgctcga ccatcacctc cagggagatc cagacggccg tgcgcctgct gctgcctggg
     3541 gagttggcca agcacgccgt gtccgagggt actaaggcca tcaccaagta caccagcgct
     3601 aagggcgggg ggtctggggg agggggatcc gggatggtga gcaagggtga ggaggataac
     3661 atggcctctc tcccagcgac tcatgagtta cacatctttg gctccatcaa cgatgtggac
     3721 tttgacatgg tgggtcaggg aaccggcaat ccaaatgaag gttatgagga gttaaacctg
     3781 aagtccacca agggcgacct ccagttctcc ccctggattc tggtccctca tatcgggtat
     3841 ggcttccatc agtacctgcc ctaccctgac gggatgtcgc ctttccaggc cgccatggta
     3901 gatggctccg gataccaagt ccatcgcaca atgcagtttg aagatggcgc ctcccttact
     3961 gttaactacc gctacaccta cgagggaagc cacatcaaag gagaggccca ggtgataggg
     4021 actggtttcc ctgctgacgg tcctgtgatg accaacacgc tgaccgctgc ggactggtgc
     4081 atgtcgaaga tgacttaccc caacgacaaa accatcatca gtacctttaa gtggagttac
     4141 atcactgtaa atggcaaacg ctaccggagc actgcgcgga ccacctacac ctttgccaag
     4201 ccaatggcgg ctaactatct gaagaaccag ccgatgtacg tgttccgtaa gacggagctc
     4261 aagcactcca tgaccgagct caacttcaag gagtggcaaa aggcctttac cgatatgatg
     4321 tgaggatccg cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat
     4381 aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg
     4441 tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc
     4501 tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt
     4561 cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg
     4621 acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac
     4681 cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg
     4741 tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg
     4801 ggcctcggta cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc
     4861 cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cgagctcatg
     4921 gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccat gagcgagctg
     4981 attaaggaga acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc
     5041 aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat gagaatcaag
     5101 gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac
     5161 ggcagcaaga ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc
     5221 cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt gctgaccgct
     5281 acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg
     5341 aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc
     5401 gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc
     5461 gtgggcggga gccatctgat cgcaaacatc aagaccacat atagatccaa gaaacccgct
     5521 aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag
     5581 gccaacaacg agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc
     5641 cctagcaaac tggggcacaa gcttaatggt ggaggtaaaa ggccggcggc cacgaaaaag
     5701 gccggccagg caaaaaagaa aaagggtgga ggtccaaaaa agaaaaggaa ggtctaagct
     5761 agcttgactg actgagatac agcgtacctt cagctcacag acatgataag atacattgat
     5821 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     5881 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     5941 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     6001 aacctctaca aatgtggtat tggcccatct ctatcggtat cgtagcataa ccccttgggg
     6061 cctctaaacg ggtcttgagg ggttttttgt gcccctcggg ccggattgct atctaccggc
     6121 attggcgcag aaaaaaatgc ctgatgcgac gctgcgcgtc ttatactccc acatatgcca
     6181 gattcagcaa cggatacggc ttccccaact tgcccacttc catacgtgtc ctccttacca
     6241 gaaatttatc cttaaggtcg tcagctatcc tgcaggcgat ctctcgattt cgatcaagac
     6301 attcctttaa tggtcttttc tggacaccac taggggtcag aagtagttca tcaaactttc
     6361 ttccctccct aatctcattg gttaccttgg gctatcgaaa cttaattaag taggtgggcg
     6421 ggccaagata ggggcgcgat tgctgcgatc tggaggacaa attacacaca cttgcgcctg
     6481 agcgccaagc acagggttgt tggtcctcat attcacgagg tcgctgagag cacggtgggc
     6541 taatgttgcc atgggtagca tatactaccc aaatatctgg atagcatatg ctatcctaat
     6601 ctatatctgg gtagcatagg ctatcctaat ctatatctgg gtagcatatg ctatcctaat
     6661 ctatatctgg gtagtatatg ctatcctaat ttatatctgg gtagcatagg ctatcctaat
     6721 ctatatctgg gtagcatatg ctatcctaat ctatatctgg gtagtatatg ctatcctaat
     6781 ctgtatccgg gtagcatatg ctatcctaat agagattagg gtagtatatg ctatcctaat
     6841 ttatatctgg gtagcatata ctacccaaat atctggatag catatgctat cctaatctat
     6901 atctgggtag catatgctat cctaatctat atctgggtag cataggctat cctaatctat
     6961 atctgggtag catatgctat cctaatctat atctgggtag tatatgctat cctaatttat
     7021 atctgggtag cataggctat cctaatctat atctgggtag catatgctat cctaatctat
     7081 atctgggtag tatatgctat cctaatctgt atccgggtag catatgctat cctcatgata
     7141 ttaattaacc agtcaagtca gctacttggc gagatcgact tgtctgggtt tcgactacgc
     7201 tcagaattgc gtcagtcaag ttcgatctgg tccttgctat tgcacccgtt ctccgattac
     7261 gagtttcatt taaatcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     7321 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     7381 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     7441 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     7501 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     7561 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     7621 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     7681 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     7741 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     7801 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     7861 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     7921 tcaa
//
