LOCUS       sequence-217036-        4092 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  General donor vector with Halo-mAID coding sequence flanking by 
            restriction sites for generating knock-in donor for Auxin inducible 
            degradation of Halo tagged endogenous protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4092)
  AUTHORS   Gu B, Posfai E, Rossant J
  TITLE     Efficient generation of targeted large insertions by microinjection 
            into two-cell-stage mouse embryos.
  JOURNAL   Nat Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166.
  PUBMED    29889212
REFERENCE   2  (bases 1 to 4092)
  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: "Nat 
            Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166."
COMMENT     Sequence Label: EasyFusion Halo-mAID
FEATURES             Location/Qualifiers
     source          1..4092
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      13..601
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     502..521
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     755..772
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    889..910
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(925..930,931..948,949..955)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    963..979
                     /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     968..990
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     987..1003
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     987..1003
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1022..1042
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1024..1042
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1071..1087
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1072..1088
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1112..1132
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             1148..2032
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     CDS             2066..2197
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Morawska and Ulrich, 
                     2013)"
                     /label=AID*
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="PKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVK"
     primer_bind     complement(2301..2320)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2302..2320)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2330..2347)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2330..2346)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2339..2361)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2487..2942)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2624..2645)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2836..2855
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2969..3073
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3074..3142,3143..3934)
                     /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     complement(3292..3311)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
       61 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
      121 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
      181 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
      241 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
      301 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
      361 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
      421 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
      481 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
      541 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
      601 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
      661 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
      721 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
      781 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
      841 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
      901 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
      961 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg
     1021 cgcaattaac cctcactaaa gggaacaaaa gctgggtacc gggccccccc tcgaggtcga
     1081 cggtatcgat aagcttgata tcgaattcat ggaggatctg tactttcaga gcgataacga
     1141 tggatccgaa atcggtactg gctttccatt cgacccccat tatgtggaag tcctgggcga
     1201 gcgcatgcac tacgtcgatg ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg
     1261 taacccgacc tcctcctacg tgtggcgcaa catcatcccg catgttgcac cgacccatcg
     1321 ctgcattgct ccagacctga tcggtatggg caaatccgac aaaccagacc tgggttattt
     1381 cttcgacgac cacgtccgct tcatggatgc cttcatcgaa gccctgggtc tggaagaggt
     1441 cgtcctggtc attcacgact ggggctccgc tctgggtttc cactgggcca agcgcaatcc
     1501 agagcgcgtc aaaggtattg catttatgga gttcatccgc cctatcccga cctgggacga
     1561 atggccagaa tttgcccgcg agaccttcca ggccttccgc accaccgacg tcggccgcaa
     1621 gctgatcatc gatcagaacg tttttatcga gggtacgctg ccgatgggtg tcgtccgccc
     1681 gctgactgaa gtcgagatgg accattaccg cgagccgttc ctgaatcctg ttgaccgcga
     1741 gccactgtgg cgcttcccaa acgagctgcc aatcgccggt gagccagcga acatcgtcgc
     1801 gctggtcgaa gaatacatgg actggctgca ccagtcccct gtcccgaagc tgctgttctg
     1861 gggcacccca ggcgttctga tcccaccggc cgaagccgct cgcctggcca aaagcctgcc
     1921 taactgcaag gctgtggaca tcggcccggg tctgaatctg ctgcaagaag acaacccgga
     1981 cctgatcggc agcgagatcg cgcgctggct gtctactctg gagatttccg gtggtagcgg
     2041 cagcggtagc actaagagtg cttgtcctaa agatccagcc aaacctccgg ccaaggcaca
     2101 agttgtggga tggccaccgg tgagatcata ccggaagaac gtgatggttt cctgccaaaa
     2161 atcaagcggt ggcccggagg cggcggcgtt cgtgaaggta tcaatggacg gagcaccgta
     2221 cttgaggaaa atcgatttga ggatgtataa agctagcact agttctagag cggccgccac
     2281 cgcggtggag ctccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     2341 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2401 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2461 agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt aatattttgt taaaattcgc
     2521 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2581 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2641 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2701 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2761 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2821 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2881 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2941 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3001 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3061 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     3121 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     3181 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     3241 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3301 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     3361 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3421 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3481 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3541 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3601 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3661 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3721 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3781 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3841 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3901 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3961 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     4021 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4081 cgtagaaaag at
//
