LOCUS       sequence-467766-        4961 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His-tagged HaloTag-linker-SpyCatcher3 
            separated by a TEV protease recognition site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4961)
  AUTHORS   Zolboot N, Xiao Y, Du JX, Ghanem MM, Choi SY, Junn MJ, Zampa F, 
            Huang Z, MacRae IJ, Lippi G
  TITLE     MicroRNA mechanisms instructing Purkinje cell specification.
  JOURNAL   Neuron. 2025 Apr 1:S0896-6273(25)00181-3. doi: 
            10.1016/j.neuron.2025.03.009.
  PUBMED    40179877
REFERENCE   2  (bases 1 to 4961)
  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: "Neuron. 
            2025 Apr 1:S0896-6273(25)00181-3. doi: 
            10.1016/j.neuron.2025.03.009."
COMMENT     Sequence Label: pET23a-His-TEV-HaloTag-linker-SpyCatcher3
FEATURES             Location/Qualifiers
     source          1..4961
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     1716..1735
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1716..1734
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1766..1788
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1808..1825
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1826..1846
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1853..2740
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="AEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSY
                     VWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIH
                     DWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIID
                     QNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVE
                     EYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLI
                     GSEIARWLSTLEISG"
     CDS             2765..3103
                     /codon_start=1
                     /product="improved SpyCatcher variant that reacts with 
                     SpyTag003 at a rate approaching the diffusion limit (Keeble
                     et al., 2019)"
                     /label=SpyCatcher003
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDG
                     EATEGDAHT"
     CDS             3177..3194
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3247..3265)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3261..3308
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3345..3800
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3432..3451)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3642..3663
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3826..3930
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3931..3999,4000..4791)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4149..4168)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     1681 acccaacgct gcccgagatc tcgatcccgc gaaattaata cgactcacta tagggagacc
     1741 acaacggttt ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatggc
     1801 tagcagccat catcatcatc atcatgagaa tctctacttc cagggtaccg gcgcagaaat
     1861 cggtactggc tttccattcg acccccatta tgtggaagtc ctgggcgagc gcatgcacta
     1921 cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta acccgacctc
     1981 ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg acccatcgct gcattgctcc
     2041 agacctgatc ggtatgggca aatccgacaa accagacctg ggttatttct tcgacgacca
     2101 cgtccgcttc atggatgcct tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat
     2161 tcacgactgg ggctccgctc tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa
     2221 aggtattgca tttatggagt tcatccgccc tatcccgacc tgggacgaat ggccagaatt
     2281 tgcccgcgag accttccagg ccttccgcac caccgacgtc ggccgcaagc tgatcatcga
     2341 tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt
     2401 cgagatggac cattaccgcg agccgttcct gaatcctgtt gaccgcgagc cactgtggcg
     2461 cttcccaaac gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga
     2521 atacatggac tggctgcacc agtcccctgt cccgaagctg ctgttctggg gcaccccagg
     2581 cgttctgatc ccaccggccg aagccgctcg cctggccaaa agcctgccta actgcaaggc
     2641 tgtggacatc ggcccgggtc tgaatctgct gcaagaagac aacccggacc tgatcggcag
     2701 cgagatcgcg cgctggctgt cgacgctcga gatttccggc ggtagcgggg aaagtggtag
     2761 cggcgtaacc accttatcag gtttatcagg tgagcaaggt ccgtccggtg atatgacaac
     2821 tgaagaagat agtgctaccc atattaaatt ctcaaaacgt gatgaggacg gccgtgagtt
     2881 agctggtgca actatggagt tgcgtgattc atctggtaaa actattagta catggatttc
     2941 agatggacat gtgaaggatt tctacctgta tccaggaaaa tatacatttg tcgaaaccgc
     3001 agcaccagac ggttatgagg tagcaactcc aattgaattt acagttaatg aggatggtca
     3061 ggttactgta gatggcgaag caactgaggg tgacgctcat actggatcca gtggtagcta
     3121 agccatggat ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc
     3181 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     3241 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     3301 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     3361 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3421 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3481 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3541 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3601 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3661 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3721 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3781 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3841 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3901 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3961 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4021 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4081 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4141 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4201 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4261 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4321 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4381 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4441 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     4501 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4561 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4621 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4681 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4741 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4801 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4861 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4921 cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
