LOCUS       sequence-440078-        8199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of MBP:6xHIS:FBF-2(121-632).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8199)
  AUTHORS   Carrick BH, Crittenden SL, Chen F, Linsley M, Woodworth J, 
            Kroll-Conner P, Ferdous AS, Keles S, Wickens M, Kimble J
  TITLE     PUF partner interactions at a conserved interface shape the 
            RNA-binding landscape and cell fate in Caenorhabditis elegans.
  JOURNAL   Dev Cell. 2024 Mar 11;59(5):661-675.e7. doi: 
            10.1016/j.devcel.2024.01.005. Epub 2024 Jan 29.
  PUBMED    38290520
REFERENCE   2  (bases 1 to 8199)
  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"; doi: 
            "10.1016/j.devcel.2024.01.005"; journalName: "Dev Cell"; date: 
            "2024-03-11- 11"; volume: "59"; issue: "5"; pages: "661-675.e7"
COMMENT     Sequence Label: pHMTc-FBF-2 (121-632)
FEATURES             Location/Qualifiers
     source          1..8199
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..875
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(801..820)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     961..983
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(977..1168)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        1664..1741
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             1742..2824
                     /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(1761..1780)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(3067..3072,3073..3088,3089..3095)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3103..3119
                     /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     3108..3130
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             3192..3209
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3210..4310
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     4284..4307
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4365..4385
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(5951..5968)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5951..5967)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5960..5982)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6146..6163)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      6316..6402
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6494..6521
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        6540..6631
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6632..6700,6701..7492)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6850..6869)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(7534..8047)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7729..7750)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7941..7960
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      join(8158..8199,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 catatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
      841 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
      901 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
      961 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     1021 ggtaaagctc atcagcgtgg tcgtgcagcg attcacagat gtctgcctgt tcatccgcgt
     1081 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     1141 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     1201 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     1261 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     1321 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     1381 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     1441 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     1501 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     1561 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     1621 tcatgcgcac ccgtggccag gacccaacgc tgcccgaaat tccgacacca tcgaatggtg
     1681 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     1741 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     1801 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     1861 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     1921 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     1981 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     2041 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     2101 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     2161 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     2221 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     2281 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     2341 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     2401 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     2461 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     2521 cgggctgcgc gttggtgcgg atatctcagt agtgggatac gacgataccg aagacagctc
     2581 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     2641 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     2701 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     2761 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     2821 gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcacaattc tcatgtttga
     2881 cagcttatca tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc
     2941 tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca aggcgcactc
     3001 ccgttctgga taatgttttt tgcgccgaca tcataacggt tctggcaaat attctgaaat
     3061 gagctgttga caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat
     3121 ttcacacagg aaacagccag tccgtttagg tgttttcacg agcacttcac caacaaggac
     3181 catagcatat gcatcatcat catcatcata tgaaaatcga agaaggtaaa ctggtaatct
     3241 ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag
     3301 ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg
     3361 ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct
     3421 acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt
     3481 atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg
     3541 ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca aaaacctggg
     3601 aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca
     3661 acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca
     3721 agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag
     3781 cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt
     3841 actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt
     3901 gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta ctgccgacct
     3961 tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca
     4021 gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact gatgaaggtc
     4081 tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag
     4141 agttggcgaa agatccacgt attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca
     4201 tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg
     4261 ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact aattcgagct
     4321 cgaacaacaa caacaataac aataacaaca acctcgggat cgaggaaaac ctgtattttc
     4381 agggcgaatt cttttcacaa aatgtccgat gtttctctgc ggatattgat cgtagcaaga
     4441 gctatggttc ttcaaaacac taccatttga aatattcgag acccgctctg tcgaggaact
     4501 ctagatcatt cacaagatca aacaatgtgc tgccaacctg gtccttggat tccaatggtg
     4561 aaatgcgttc aagattgtcg ctctctgaag ttttggatag tggcgattta atgaagtttg
     4621 ccgttgacaa aactgggtgc cagttcctag aaaaggcagt gaagggcagc ctgacatctt
     4681 atcagaagtt ccaattgttt gaacaagtta ttgggagaaa agatgatttc cttaaattga
     4741 gcacgaacat ctttggaaat tatttggtac aaagcgttat tggaatttca ttggcaacta
     4801 atgatgacgg atacacaaag cgacaagaaa agttgaagaa cttcatcagt tcccaaatga
     4861 cagatatgtg cttggacaaa tttgcgtgtc gtgttatcca atccagtctc cagaacatgg
     4921 atttatccct tgcgtgtaaa cttgttcaag ctctacctcg tgatgctcgt cttatcgcaa
     4981 tctgtgttga tcaaaatgcc aatcacgtca ttcagaaagt cgtagcagtc atcccactga
     5041 agaattggga gtttattgtg gatttcgttg ccaccccaga gcacctccgt cagatttgtt
     5101 ctgataagta tgggtgccgt gttgtgcaaa ctattatcga aaagctcact gctgattcaa
     5161 tgaacgttga tttaacttcc gctgctcaaa atcttcgtga acgagcgctc caacgactca
     5221 tgacttctgt gactaatcgg tgccaagaat tagctacgaa cgagtatgcc aattacataa
     5281 tacagcatat tgtatccaat gatgatctgg ccgtttatcg ggaatgtatc atagagaaat
     5341 gcctcatgcg caaccttctt tcgttgtccc aggaaaagtt tgcttctcat gttgttgaga
     5401 aagcattcct acatgcacca ttggaacttc ttgccgaaat gatggacgag attttcgacg
     5461 gatacattcc acatccggac acgggaaaag atgccctcga tatcatgatg ttccatcaat
     5521 tcggaaacta cgtcgttcaa tgcatgctga ctatttgctg tgatgctgtg tctggaagac
     5581 ggcaaacgaa agaaggaggc tatgaccatg caatctcatt ccaggattgg ctgaaaaagt
     5641 tgcactcgag agttacgaaa gaaaggcatc gcttgtcaag gttctcatcc ggcaaaaaga
     5701 tgatcgagac gcttgccaat ctccgttcaa ctcatccgat ttatgaactc caatcttcgg
     5761 gacacgattc attcaagacg gattacttca gtactgcttc cgagcacgat ggtccggagt
     5821 tggagaagaa tgggatcgag gaaggaagcc tgatgctaga gccacggagc aataagtctt
     5881 cagtctctgt caaattttct tcttccggta gtcacggcga tgattaagtc gacctgcagg
     5941 caagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccca ggcgttaccc
     6001 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     6061 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcag cttggctgtt
     6121 ttggcggatg agataagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc
     6181 tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga
     6241 actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag
     6301 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     6361 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg
     6421 aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg
     6481 catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt
     6541 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     6601 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     6661 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     6721 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     6781 cagcggtaag atccttgaga gttttcgccc cgaagaacgt ttcccaatga tgagcacttt
     6841 taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
     6901 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     6961 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     7021 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     7081 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     7141 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     7201 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     7261 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     7321 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     7381 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     7441 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     7501 ccaagtttac tcatatatac tttagattga tttaccccgg ttgataatca gaaaagcccc
     7561 aaaaacagga agattgtata agcaaatatt taaattgtaa acgttaatat tttgttaaaa
     7621 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     7681 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     7741 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     7801 ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttggggtc gaggtgccgt
     7861 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     7921 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     7981 agtgtagcgg tcacgctgcg cgtaaacacc acacccgccg cgcttaatgc gccgctacag
     8041 ggcgcgtaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     8101 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     8161 agatcctttt tttctgcgcg taatctgctg cttgcaaac
//
