LOCUS       sequence-438224-        6832 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repair template to endogenously tag SMC4 with mAID-Halo Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6832)
  AUTHORS   Batty P, Langer CC, Takacs Z, Tang W, Blaukopf C, Peters JM, Gerlich
            DW
  TITLE     Cohesin-mediated DNA loop extrusion resolves sister chromatids in G2
            phase.
  JOURNAL   EMBO J. 2023 Aug 15;42(16):e113475. doi: 10.15252/embj.2023113475. 
            Epub 2023 Jun 26.
  PUBMED    37357575
REFERENCE   2  (bases 1 to 6832)
  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: "EMBO J."; 
            date: "2023-08-15"; volume: "42(16):e113475. doi"; pages: " 
            10.15252/embj.2023113475. Epub 2023 Jun 26"
COMMENT     Sequence Label: RepairTemplate_Smc4-mAID-HaloTAG
FEATURES             Location/Qualifiers
     source          1..6832
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(96..983)
                     /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             complement(1005..1208)
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             complement(1212..1229)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1239..1256)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2179..2195)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2179..2195)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2192..2214)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2203..2219
                     /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)."
     promoter        complement(join(2227..2233,2234..2251,2252..2257))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2272..2293
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2410..2427)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2581..3169)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2661..2680)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3340..4131,4132..4200))
                     /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     3963..3982
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(4218..5012)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4329..4348)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4939..4958
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      complement(5356..5784)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5466..5487)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5678..5697
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5910..5932
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5924..5941
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5925..5941
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5948..5967
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5948..5966
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 tgtattttat acaactcata atcctttaca aatagaaaaa aatcagtaat acactgaatc
       61 aacttgaaga atcttcagca taaagtacgc gttcagccgg aaatctcgag cgtcgacagc
      121 cagcgcgcga tctcgctgcc gatcaggtcc gggttgtctt cttgcagcag attcagaccc
      181 gggccgatgt ccacagcctt gcagttaggc aggcttttgg ccaggcgagc ggcttcggcc
      241 ggtgggatca gaacgcctgg ggtgccccag aacagcagct tcgggacagg ggactggtgc
      301 agccagtcca tgtattcttc gaccagcgcg acgatgttcg ctggctcacc ggcgattggc
      361 agctcgtttg ggaagcgcca cagtggctcg cggtcaacag gattcaggaa cggctcgcgg
      421 taatggtcca tctcgacttc agtcagcggg cggacgacac ccatcggcag cgtaccctcg
      481 ataaaaacgt tctgatcgat gatcagcttg cggccgacgt cggtggtgcg gaaggcctgg
      541 aaggtctcgc gggcaaattc tggccattcg tcccaggtcg ggatagggcg gatgaactcc
      601 ataaatgcaa tacctttgac gcgctctgga ttgcgcttgg cccagtggaa acccagagcg
      661 gagccccagt cgtgaatgac caggacgacc tcttccagac ccagggcttc gatgaaggca
      721 tccatgaagc ggacgtggtc gtcgaagaaa taacccaggt ctggtttgtc ggatttgccc
      781 ataccgatca ggtctggagc aatgcagcga tgggtcggtg caacatgcgg gatgatgttg
      841 cgccacacgt aggaggaggt cgggttaccg tgcaggaaca gcacaggggt gccatcgcgc
      901 ggaccaacat cgacgtagtg catgcgctcg cccaggactt ccacataatg ggggtcgaat
      961 ggaaagccag taccgatttc tgcgatatct ccacctccac ctcccttata catcctcaaa
     1021 tcgattttcc tcaagtatgg tgctccgtcc attgatacct tcacgaatgc agctgcctct
     1081 ggtccaccgc ttgatttttg gcaggaaacc atcacgttct tccggtatga tctcaccggt
     1141 ggccatccca caacttgtgc cttggccgga ggtttggctg gatctttagg acaagcactc
     1201 ttctccttat ggctgccgcg cggcaccagg ccgctgctat gatggtgatg atggtgactg
     1261 ctcgtcgact gcagaattcg aagcttgagc tcgagatctg agtaacaaag tccttttgac
     1321 gctatctcct tcggatttac agcaacactt tttgttatgt tgtatgtctt gtaaattcca
     1381 ataagtctat ccgaaatctc aaacatatta tttcgaagag aaattattat gaactgtgca
     1441 ttttttgttt gttcctaaag agaaacaaag aaaagtcaaa cattaagtaa taacaggtaa
     1501 atcacacaaa catgtaacct caaatatttt ttggacaata aacattacat tgataacatg
     1561 taaaggaatg atggcaggaa taattcataa aacagtattt gagtgtgaaa ggaatttggt
     1621 tattttgact tctagtggaa aagatctagg aagatgacct aggattctag ctgtgattct
     1681 gctactaatt tgcattttta atttatattt tactcctcca aaatggaaat atttggttca
     1741 aataaaaaat aatttggtct ccactgatac cagaggagat ggagatggag ttattagaca
     1801 ataaagaaaa agctcaattg taagaaataa agaattccat gtgtaatgat taggataccc
     1861 ctaaaagttt ctggaatgaa aatcccaaaa tgattactta catatatata aaatgcaaca
     1921 atggacacat ttttaaaatc aagggctgca tcaatctcat ccatgaagta aaggggagtg
     1981 ggcttgtagt ggtgaagagc aaatactaaa gccaatgaac taagtgtttt ctctcctccc
     2041 gaaaggttga agatcttttt ccaacttttc ttaggtggtc gaacactgaa atgattaaga
     2101 aaatatctgc agaattccag cacactggcg gccgttacta gtggatccga gctcggtacc
     2161 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     2221 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     2281 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2341 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     2401 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2461 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2521 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2581 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2641 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2701 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2761 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2821 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2881 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2941 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3001 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3061 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3121 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3181 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     3241 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     3301 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     3361 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     3421 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     3481 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3541 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3601 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     3661 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     3721 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     3781 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     3841 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     3901 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     3961 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4021 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4081 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     4141 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     4201 actcttcctt tttcaattca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc
     4261 gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc
     4321 tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc
     4381 cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag
     4441 gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg
     4501 aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga
     4561 ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg
     4621 caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc
     4681 tcggcaggag caaggtggga tgacaggaga tcctgccccg gcacttcgcc caatagcagc
     4741 cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg
     4801 gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg
     4861 gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag
     4921 cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga
     4981 gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga
     5041 tcagatcttg atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact
     5101 ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccgg ttcgcttgct
     5161 gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt
     5221 ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt
     5281 cagcaccgtt tctgcggact ggctttctac gtgttccgct tcctttagca gcccttgcgc
     5341 cctgaatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     5401 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     5461 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     5521 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     5581 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     5641 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     5701 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     5761 ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact gttgggaagg
     5821 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     5881 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
     5941 tgaattgtaa tacgactcac tatagggcga attgggccct ctagatgcat gctcgagcgg
     6001 ccgccagtgt gatggatcct tagcattata acattcttga gccaatagtg taaaaatacg
     6061 ctgacaattt tataggcaaa cattactcaa ggtatcttac tttccactta ttactaaagt
     6121 aattaacccc taaatagatg ctcctcaaca gtgggactac atcctggtaa acctatcata
     6181 agttgaaact atcaagttga aatgcattta gtacccggat aaacctatca taaagttgaa
     6241 aatttgtaaa ttgaaccagt gtaaatcaga ggccatctta cttcatactc atgaagcaac
     6301 tatagtggga tatttttcaa cttacgagat agcctaggct tgttgaaaca ctgtcctaat
     6361 ttactggctc tctggtaatt aagtcataaa tggtcaaaca tcaaattcta gaaaagcata
     6421 tatttaaatt ctcatcacta taattcagaa tgactggttt acaaatatat gttcatagta
     6481 tttgggtaat aactgtggtg attgtctcaa ctggcaagct aagtggaatg ccttaaagaa
     6541 tctatccaag attagttggc aggcatttcc agtgttggta aatcaaaggc ccacgaaggt
     6601 atgctgagcc ttgatagcac aaccttgctt aggactttgc ctcaactaac atacttttta
     6661 gtttggcaag ttacctgacc ctccaggtaa ctcatttttc tccatagtct gctacttaca
     6721 agattgtcat atttttgtaa tctagaagca attatagaga atattttatt agactttaca
     6781 aatgacaact gcataaaaca gaaaccagtt tcatgatcta gtttagggag ta
//
