LOCUS       sequence-409948-        6194 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Parent vector for producing universal minicircle donors for HaloTag 
            C-terminal knock-in.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6194)
  AUTHORS   Shi Y, Kopparapu N, Ohler L, Dickinson DJ
  TITLE     Efficient and rapid fluorescent protein knock-in with universal 
            donors in mouse embryonic stem cells.
  JOURNAL   Development. 2023 May 15;150(10):dev201367. doi: 10.1242/dev.201367.
            Epub 2023 May 25.
  PUBMED    37129004
REFERENCE   2  (bases 1 to 6194)
  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: 
            "Development."; date: "2023-05-15"; volume: "150(10):dev201367. 
            doi"; pages: " 10.1242/dev.201367. Epub 2023 May 25"
COMMENT     Sequence Label: pYS28
FEATURES             Location/Qualifiers
     source          1..6194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(785..802)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    823..856
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             943..1827
                     /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             1840..1893
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1894..2916
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(3059..3853)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3059..3853)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3170..3189)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3780..3799
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4217..4234)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4387..4473
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4565..4592
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4726..5314
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5215..5234
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5468..5485
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5500..5640
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5566..5585)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5726..5748
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tcttctctca tccgccaaaa cagccaagct ggagaccgtt tgacattacc ctgttatccc
       61 tagatacatt accctgttat cccagatgac ataccctgtt atccctagat gacattaccc
      121 tgttatccca gatgacatta ccctgttatc cctagataca ttaccctgtt atcccagatg
      181 acataccctg ttatccctag atgacattac cctgttatcc cagatgacat taccctgtta
      241 tccctagata cattaccctg ttatcccaga tgacataccc tgttatccct agatgacatt
      301 accctgttat cccagatgac attaccctgt tatccctaga tacattaccc tgttatccca
      361 gatgacatac cctgttatcc ctagatgaca ttaccctgtt atcccagatg acattaccct
      421 gttatcccta gatacattac cctgttatcc cagatgacat accctgttat ccctagatga
      481 cattaccctg ttatcccaga tgacattacc ctgttatccc tagatacatt accctgttat
      541 cccagatgac ataccctgtt atccctagat gacattaccc tgttatccca gatgacatta
      601 ccctgttatc cctagataca ttaccctgtt atcccagatg acataccctg ttatccctag
      661 atgacattac cctgttatcc cagatgacat taccctgtta tccctagata cattaccctg
      721 ttatcccaga tgacataccc tgttatccct agatgacatt accctgttat cccagataaa
      781 ctcaatgatg atgatgatga tggtcgagac tcagcggccg cggtgccagg gcgtgccctt
      841 gggctccccg ggcgcgacgt gacgctgctg acccctttct ttcccttctg caggggccag
      901 tacccaaaaa gcggggggtc tggtggcagt ggagggggat ccgaaatagg tactggattt
      961 ccctttgacc cccactatgt ggaggtgctt ggggaacgaa tgcactatgt tgacgtgggt
     1021 cccagagatg ggacccccgt gttgtttttg catggaaatc caactagttc ctatgtgtgg
     1081 cgaaatataa ttccacatgt cgcacctacc caccgctgta ttgcccccga cctcattggt
     1141 atgggaaaat ctgataagcc agatttggga tatttctttg atgaccatgt gaggtttatg
     1201 gacgccttca ttgaagcctt ggggcttgag gaagtagtgc tcgtaattca cgattggggg
     1261 agcgctctgg gttttcactg ggccaagagg aacccagaaa gggttaaggg tatcgcattt
     1321 atggagttca tccgcccaat cccaacttgg gatgaatggc ctgagtttgc ccgcgagaca
     1381 ttccaggcct ttaggactac cgatgttgga cggaagttga ttatcgatca gaacgtgttt
     1441 atagagggaa ctttgccaat gggggtggta aggcccctga cagaagtgga aatggatcat
     1501 tatagggagc ccttcttgaa tcctgtagac agggaacccc tgtggagatt ccccaacgaa
     1561 ttgcccattg caggtgagcc tgccaatatc gtagcactcg tcgaggaata catggattgg
     1621 ttgcatcaaa gtcccgtccc aaaacttttg ttctggggaa cccctggtgt tctcattcca
     1681 ccagcagagg cagctcggtt ggcaaaatca cttccaaatt gcaaagcagt agatatcggg
     1741 cctggcctga acctgttgca ggaggacaac ccagatttga taggctcaga aatagcccga
     1801 tggctgtcaa ctttggagat tagcggtggt tccggaggag agggcagagg aagtctgcta
     1861 acatgcggtg acgtcgagga gaatcctggc ccaatgaaaa agcccgaact tacagctacc
     1921 agtgtggaga aatttcttat tgaaaaattc gacagcgtat cagatctgat gcaacttagc
     1981 gaaggagagg aaagcagagc ttttagcttt gacgtgggcg gacgagggta tgtactgcgc
     2041 gtcaatagtt gtgcagatgg cttttacaag gatcggtacg tctaccggca ctttgcatca
     2101 gctgctttgc ctatccctga agtccttgac ataggagagt tcagtgagag tctcacttac
     2161 tgcatttcac gcagagcaca aggtgtgaca ctccaagacc tccctgaaac cgagttgccc
     2221 gccgttcttc aaccagtcgc cgaggcaatg gatgctatag cagcagcaga tcttagccag
     2281 accagcggct tcggtccatt cggcccacag ggaatcggtc agtacacaac ttggcgagat
     2341 tttatttgcg ctatagcaga cccccacgtc tatcactggc agacagtaat ggacgacact
     2401 gtgtcagcct ctgttgctca agctttggat gagcttatgc tgtgggctga ggattgtcca
     2461 gaagttcgcc acctcgtgca tgctgatttc ggttctaata atgtacttac agataatggc
     2521 aggataactg cagtcataga ctggagcgaa gcaatgtttg gagactccca gtatgaggtc
     2581 gcaaatattt ttttttggag gccatggctc gcctgtatgg aacaacagac tcgatacttt
     2641 gaacggcgcc accctgagct tgctggttct ccacggctcc gggcctacat gctgaggata
     2701 gggctcgatc aattgtacca atcattggtg gacggcaatt ttgatgacgc tgcttgggct
     2761 caaggtagat gtgacgctat cgtccggtcc ggtgcaggga ccgtaggacg gactcagata
     2821 gctagacgca gtgccgctgt atggactgat ggttgtgttg aagtgcttgc cgactccggc
     2881 aaccggcggc cttccacacg gccccgggct aaagaagtaa gtcgactcgt tcgccccaac
     2941 tggggtaacc tttgagttct ctcagttggg ggtaatcagc atcatgatgt ggtaccacat
     3001 catgatgctg attataagaa tgcggccgcc acactctagt ggatctcgag ttaataattc
     3061 agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac
     3121 cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg
     3181 tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc
     3241 cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga
     3301 cgagatcctc gccgtcgggc atgctcgcct tgagcctggc gaacagttcg gctggcgcga
     3361 gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac
     3421 gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg
     3481 tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag
     3541 atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag
     3601 tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg
     3661 ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg
     3721 ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg
     3781 cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat
     3841 cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagagctt gatcccctgc
     3901 gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc ttcccaacct
     3961 taccagaggg cgccccagct ggcaattccg gttcgcttgc tgtccataaa accgcccagt
     4021 ctagctatcg ccatgtaagc ccactgcaag ctacctgctt tctctttgcg cttgcgtttt
     4081 cccttgtcca gatagcccag tagctgacat tcatccgggg tcagcaccgt ttctgcggac
     4141 tggctttcta cgcgctcgag ggggggccaa acggtctcca gcttggctgt tttggcggat
     4201 gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac
     4261 agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag
     4321 tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc
     4381 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     4441 ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg
     4501 aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt
     4561 aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct tttgtttatt
     4621 tttctaaata cattcaaata tgtatccgct catgaccaaa atcccttaac gtgagttttc
     4681 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4741 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4801 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4861 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4921 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4981 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5041 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5101 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5161 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5221 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5281 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5341 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5401 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     5461 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     5521 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
     5581 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     5641 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5701 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5761 tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcataa
     5821 tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc tatgctactc cgtcaagccg
     5881 tcaattgtct gattcgttac caattatgac aacttgacgg ctacatcatt cactttttct
     5941 tcacaaccgg cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag
     6001 aaatagagtt gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg
     6061 gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta
     6121 atccctaact gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt
     6181 gcgacgctgg cgat
//
