LOCUS       sequence-474937-        7120 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-overexpression of Rhobin2-SEC61B 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 7120)
  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 7120)
  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-SEC61B-IRES-NLS-mTagBFP-2xNLS
FEATURES             Location/Qualifiers
     source          1..7120
                     /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    3527..4100
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3550..4102
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3694..3711)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3921..3940
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4120..4140
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4165..4863
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             4873..4920
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4930..4950
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    5003..5124
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5014..5033)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5068..5087
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     terminator      5239..5285
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      5315..5451
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    5496..5567
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(6532..7120)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6612..6631)
                     /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 caggtggcat gccaggccca
     3241 accccatccg gcacaaatgt tgggtcaagc ggccgtagcc ctagtaaagc agtggccgca
     3301 cgcgccgccg gaagtaccgt gagacagcgc aaaaacgcat catgtggaac gcgctccgcc
     3361 gggaggacca cgtctgctgg gacagggggc atgtggaggt tctacaccga ggactctccg
     3421 ggactcaagg tgggtccagt tccggtgctc gtcatgtccc tgctgttcat tgcctccgtt
     3481 ttcatgcttc acatatgggg caaatacaca cgctcctgag gatccgcccc tctccctccc
     3541 ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata
     3601 tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg
     3661 tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt
     3721 tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag
     3781 cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc
     3841 cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga
     3901 tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg
     3961 cccagaaggt accccattgt atgggatctg atctggggcc tcggtacaca tgctttacat
     4021 gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc
     4081 tttgaaaaac acgatgataa tatggccgag ctcatggccc caaagaagaa gcggaaggtc
     4141 ggtatccacg gagtcccagc agccatgagc gagctgatta aggagaacat gcacatgaag
     4201 ctgtacatgg agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc
     4261 aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc
     4321 ttcgccttcg acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac
     4381 acccagggca tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga
     4441 gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac
     4501 ggctgcctca tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg
     4561 atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc
     4621 ggcctggaag gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca
     4681 aacatcaaga ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc
     4741 tactatgtgg actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag
     4801 cagcacgagg tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctt
     4861 aatggtggag gtaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag
     4921 ggtggaggtc caaaaaagaa aaggaaggtc taagctagct tgactgactg agatacagcg
     4981 taccttcagc tcacagacat gataagatac attgatgagt ttggacaaac cacaactaga
     5041 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     5101 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     5161 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtattggc
     5221 ccatctctat cggtatcgta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     5281 ttttgtgccc ctcgggccgg attgctatct accggcattg gcgcagaaaa aaatgcctga
     5341 tgcgacgctg cgcgtcttat actcccacat atgccagatt cagcaacgga tacggcttcc
     5401 ccaacttgcc cacttccata cgtgtcctcc ttaccagaaa tttatcctta aggtcgtcag
     5461 ctatcctgca ggcgatctct cgatttcgat caagacattc ctttaatggt cttttctgga
     5521 caccactagg ggtcagaagt agttcatcaa actttcttcc ctccctaatc tcattggtta
     5581 ccttgggcta tcgaaactta attaagtagg tgggcgggcc aagatagggg cgcgattgct
     5641 gcgatctgga ggacaaatta cacacacttg cgcctgagcg ccaagcacag ggttgttggt
     5701 cctcatattc acgaggtcgc tgagagcacg gtgggctaat gttgccatgg gtagcatata
     5761 ctacccaaat atctggatag catatgctat cctaatctat atctgggtag cataggctat
     5821 cctaatctat atctgggtag catatgctat cctaatctat atctgggtag tatatgctat
     5881 cctaatttat atctgggtag cataggctat cctaatctat atctgggtag catatgctat
     5941 cctaatctat atctgggtag tatatgctat cctaatctgt atccgggtag catatgctat
     6001 cctaatagag attagggtag tatatgctat cctaatttat atctgggtag catatactac
     6061 ccaaatatct ggatagcata tgctatccta atctatatct gggtagcata tgctatccta
     6121 atctatatct gggtagcata ggctatccta atctatatct gggtagcata tgctatccta
     6181 atctatatct gggtagtata tgctatccta atttatatct gggtagcata ggctatccta
     6241 atctatatct gggtagcata tgctatccta atctatatct gggtagtata tgctatccta
     6301 atctgtatcc gggtagcata tgctatcctc atgatattaa ttaaccagtc aagtcagcta
     6361 cttggcgaga tcgacttgtc tgggtttcga ctacgctcag aattgcgtca gtcaagttcg
     6421 atctggtcct tgctattgca cccgttctcc gattacgagt ttcatttaaa tcatgtgagc
     6481 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     6541 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     6601 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     6661 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     6721 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     6781 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     6841 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     6901 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     6961 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     7021 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     7081 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
//
