LOCUS       sequence-472824-        5693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial overexpression of de novo Rhodamine binder 2 (Rhobin2) 
            protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5693)
  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 5693)
  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: pET29b-Rhobin2
FEATURES             Location/Qualifiers
     source          1..5693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     32..51
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        32..50
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    51..75
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             90..112
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             123..140
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             141..161
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(655..673)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      669..716
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      753..1208
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(840..859)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1050..1071
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1300..2115)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1300..2115)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2023..2042
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2107..2126
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2237..2825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2726..2745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2979..2996
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3011..3150
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3076..3095)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3236..3258
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3252..3443)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4252..5334)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5296..5315
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5335..5412)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5618..5637
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aggatcgaga tcgatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga
       61 gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga gatatacata
      121 tgcaccacca ccaccaccac gaaaacctgt acttccagag cgagaaaaat cagtttatca
      181 tcgatattta tgaaaagttg aatgagtacc acaagaagct gctcgaactg gcgagagcgc
      241 tggcagaagc acttcgggcc ggggatcgag cgcgcgctgg ccgattgctg gaggagttcc
      301 ttgcgacgtt caaagcaatg aaagcatacg gagatgctaa agaggtcgag agttttctct
      361 acttcagtga ggaggaactt gcggccatat ttgcggaatc agatgtcgaa cggaagaagc
      421 tgggagtgac actcaagatc cctgaagtcg tggccatggt agaggaactg ggtcgcgcat
      481 ttgaggctgg ggatactgct actgcggcgg cgatagtaga aaagttggta aagttttacg
      541 aggcaaacat aataataggc caaaagagaa tcgatgaact caagtcaaaa atttaactcg
      601 agtgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
      661 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
      721 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
      781 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
      841 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
      901 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
      961 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1021 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1081 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     1141 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     1201 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1261 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     1321 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     1381 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     1441 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     1501 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     1561 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     1621 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     1681 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     1741 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     1801 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     1861 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     1921 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     1981 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     2041 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     2101 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     2161 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2221 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2281 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2341 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     2401 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2461 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2521 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2581 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2641 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2701 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2761 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2821 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2881 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     2941 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3001 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3061 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     3121 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     3181 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3241 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     3301 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     3361 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     3421 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     3481 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     3541 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     3601 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     3661 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     3721 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     3781 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     3841 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     3901 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     3961 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     4021 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     4081 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     4141 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4201 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4261 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4321 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     4381 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     4441 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     4501 gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc
     4561 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     4621 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     4681 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     4741 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     4801 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     4861 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     4921 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     4981 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     5041 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     5101 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     5161 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     5221 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     5281 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     5341 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     5401 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     5461 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     5521 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     5581 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     5641 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tag
//
