LOCUS       sequence-477745-        6374 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a de novo dimer fused to FRB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6374)
  AUTHORS   Gregorio NE, Li Z, Hoye JW, David Baker D, DeForest CA
  TITLE     Stimuli-triggered formation of de novo-designed protein biomaterials
  JOURNAL   Cell Biomaterials, 2025
REFERENCE   2  (bases 1 to 6374)
  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: "Cell 
            Biomaterials, 2025"
COMMENT     Sequence Label: Dimer-FRB
FEATURES             Location/Qualifiers
     source          1..6374
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(16..63)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     59..77
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(130..147)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1267..1284)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1301..1323
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1338..1362
                     /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)."
     primer_bind     complement(1362..1381)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1363..1381)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1469..1488)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1694..1771
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1772..2854
                     /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     complement(1791..1810)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3663..3854
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3848..3870)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3956..4095
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4011..4030
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4110..4127)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4281..4869)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4361..4380)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4980..4999)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4991..5806
                     /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             4991..5806
                     /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     complement(5064..5083)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5898..6353)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6035..6056)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6247..6266
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc
       61 tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt tgttagcagc
      121 cggatctcag tggtggtggt ggtggtgctc gagttatttg ctaatgcggc gaaacacatg
      181 ataatacaga tcccacgcct gggtcagatc tttaacgtta ccgcttttca tatatttgcg
      241 gcaccattcc tgcgcttcca tcagatcgcg gccataagcc tggttaaagc tggtttcttt
      301 cagcgtctgc gggccgcgtt ccatcatcgc atgcagcggt tccagcactt caaacatgcc
      361 tttcacgttg cgttcgccaa aatacagacg gcttgcttct tccagacctt cgtgccacat
      421 ttcatgccac agaatgcttc ctccgctccc accgctacca ccactaccgc ctgaaccgcc
      481 ttcttttgct tcccgaatta gttcttccgc gcgtttgacc agctcacggc attcgtcttc
      541 atcagtgctt tctttgcact gttctatcag ttctttggct tctttaagcg cacgtttcgc
      601 ttcttcggaa tctggataac gtttggcgag ttcaatcaat ttctgcgcta atttcaccag
      661 ttcttcacat aactcttcat ccgtgctttg tttgcataat tcgatcgcct ccagggcggc
      721 tttcaacgcg agttttgcag cttcgctgtc cggatagcgt ttcgccagct cgatcagttt
      781 ctgagccagt ttaaccagtt cttcgcacaa ttcttcgtcg gtgctctgtt tgcaaagttc
      841 gatggcttcc agcgccgctt tcagtgcaag tttcgcggct tcgctatccg gatacagcag
      901 tgccagttcc ataagcagca gggccaggcg caacagcagc tggcacagtt cttcatcggt
      961 actgcgtttg cacagctcaa tcgcttcacg aatcgcacga agtgccaatt cagccgcttc
     1021 gctacgaata gccgccagca gcagttccgc gatcagcaga atttgttccg caatcagttt
     1081 caggatttcg cgcactaatt cttttatttc cctcagcagc tcgcgcacat cttcatctga
     1141 gctaccttcg cgctgcagcc tttccagttc acgcagcaga cgttccagtc tagcagtcaa
     1201 ttcggcctgt tccgccaggc tgcgcgccag ttcgcgaata atttcttcac gggtaccgct
     1261 gctgccatga tggtggtgat gatgaccgcg catatgtata tctccttctt aaagttaaac
     1321 aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt gagtcgtatt
     1381 aatttcgcgg gatcgagatc gatctcgatc ctctacgccg gacgcatcgt ggccggcatc
     1441 accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga tggggaagat
     1501 cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt ggcaggcccc
     1561 gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc ggcggcggtg
     1621 ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca taagggagag
     1681 cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg
     1741 cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc
     1801 agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt
     1861 ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa
     1921 ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag
     1981 tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact
     2041 gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc
     2101 ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga
     2161 ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt
     2221 ctctgaccag acacccatca acagtattat tttctcccat gaagacggta cgcgactggg
     2281 cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag
     2341 ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat
     2401 tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat
     2461 gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc
     2521 gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg acatctcggt
     2581 agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa
     2641 acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg
     2701 ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct
     2761 ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     2821 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc
     2881 tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac ccagtcagct
     2941 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca
     3001 tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag gaccgctttc
     3061 gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg
     3121 ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag gccattatcg
     3181 ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag
     3241 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
     3301 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
     3361 gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct
     3421 gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct
     3481 ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac
     3541 cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc
     3601 tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat
     3661 cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat
     3721 taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat
     3781 cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg
     3841 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     3901 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     3961 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     4021 gcagattgta ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga
     4081 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4141 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4201 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4261 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4321 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4381 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4441 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4501 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4561 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4621 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4681 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     4741 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4801 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4861 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4921 tcacgttaag ggattttggt catgaacaat aaaactgtct gcttacataa acagtaatac
     4981 aaggggtgtt atgagccata ttcaacggga aacgtcttgc tctaggccgc gattaaattc
     5041 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     5101 tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     5161 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     5221 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg catggttact
     5281 caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     5341 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     5401 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     5461 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     5521 agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg tcactcatgg
     5581 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     5641 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     5701 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     5761 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat taattcatga
     5821 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5881 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     5941 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     6001 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     6061 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     6121 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     6181 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     6241 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     6301 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     6361 gccaatccgg atat
//
