LOCUS       sequence-217031-        3885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  General donor vector with HaloTag coding sequence flanking by 
            restriction sites for generating knock-in donor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3885)
  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 3885)
  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
FEATURES             Location/Qualifiers
     source          1..3885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      22..610
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     511..530
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     764..781
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    898..919
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(934..939,940..957,958..964)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    972..988
                     /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     977..999
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     996..1012
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     996..1012
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1031..1051
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1033..1051
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1080..1096
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1081..1097
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1121..1141
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             1157..2041
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     primer_bind     complement(2103..2122)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2104..2122)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2132..2149)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2132..2148)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2141..2163)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2289..2744)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2426..2447)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2638..2657
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2771..2875
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2876..2944,2945..3736)
                     /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(3094..3113)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
       61 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
      121 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
      181 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
      241 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
      301 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
      361 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
      421 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
      481 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
      541 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
      601 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
      661 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
      721 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
      781 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
      841 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
      901 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
      961 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta
     1021 cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctgggtaccg ggccccccct
     1081 cgaggtcgac ggtatcgata agcttgatat cgaattcatg gaggatctgt actttcagag
     1141 cgataacgat ggatccgaaa tcggtactgg ctttccattc gacccccatt atgtggaagt
     1201 cctgggcgag cgcatgcact acgtcgatgt tggtccgcgc gatggcaccc ctgtgctgtt
     1261 cctgcacggt aacccgacct cctcctacgt gtggcgcaac atcatcccgc atgttgcacc
     1321 gacccatcgc tgcattgctc cagacctgat cggtatgggc aaatccgaca aaccagacct
     1381 gggttatttc ttcgacgacc acgtccgctt catggatgcc ttcatcgaag ccctgggtct
     1441 ggaagaggtc gtcctggtca ttcacgactg gggctccgct ctgggtttcc actgggccaa
     1501 gcgcaatcca gagcgcgtca aaggtattgc atttatggag ttcatccgcc ctatcccgac
     1561 ctgggacgaa tggccagaat ttgcccgcga gaccttccag gccttccgca ccaccgacgt
     1621 cggccgcaag ctgatcatcg atcagaacgt ttttatcgag ggtacgctgc cgatgggtgt
     1681 cgtccgcccg ctgactgaag tcgagatgga ccattaccgc gagccgttcc tgaatcctgt
     1741 tgaccgcgag ccactgtggc gcttcccaaa cgagctgcca atcgccggtg agccagcgaa
     1801 catcgtcgcg ctggtcgaag aatacatgga ctggctgcac cagtcccctg tcccgaagct
     1861 gctgttctgg ggcaccccag gcgttctgat cccaccggcc gaagccgctc gcctggccaa
     1921 aagcctgcct aactgcaagg ctgtggacat cggcccgggt ctgaatctgc tgcaagaaga
     1981 caacccggac ctgatcggca gcgagatcgc gcgctggctg tctactctgg agatttccgg
     2041 tggtagcggc agcggtagca ctagttctag agcggccgcc accgcggtgg agctccaatt
     2101 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     2161 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     2221 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     2281 gcgaatggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     2341 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     2401 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     2461 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     2521 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     2581 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     2641 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     2701 aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg
     2761 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2821 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2881 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2941 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3001 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3061 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     3121 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3181 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3241 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3301 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3361 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     3421 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3481 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3541 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3601 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3661 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3721 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3781 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3841 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgt
//
