LOCUS       sequence-392299-        6153 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Parent vector for producing universal minicircle donors for HaloTag 
            N-terminal knock-in.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6153)
  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 6153)
  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: pYS29
FEATURES             Location/Qualifiers
     source          1..6153
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(743..760)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    781..814
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             864..917
                     /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             918..1802
                     /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             1854..2876
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(3019..3813)
                     /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(3019..3813)
                     /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(3130..3149)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3740..3759
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4176..4193)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4346..4432
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4524..4551
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4685..5273
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5174..5193
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5427..5444
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5459..5599
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5525..5544)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5685..5707
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 acattaccct gttatcccta gatacattac cctgttatcc cagatgacat accctgttat
       61 ccctagatga cattaccctg ttatcccaga tgacattacc ctgttatccc tagatacatt
      121 accctgttat cccagatgac ataccctgtt atccctagat gacattaccc tgttatccca
      181 gatgacatta ccctgttatc cctagataca ttaccctgtt atcccagatg acataccctg
      241 ttatccctag atgacattac cctgttatcc cagatgacat taccctgtta tccctagata
      301 cattaccctg ttatcccaga tgacataccc tgttatccct agatgacatt accctgttat
      361 cccagatgac attaccctgt tatccctaga tacattaccc tgttatccca gatgacatac
      421 cctgttatcc ctagatgaca ttaccctgtt atcccagatg acattaccct gttatcccta
      481 gatacattac cctgttatcc cagatgacat accctgttat ccctagatga cattaccctg
      541 ttatcccaga tgacattacc ctgttatccc tagatacatt accctgttat cccagatgac
      601 ataccctgtt atccctagat gacattaccc tgttatccca gatgacatta ccctgttatc
      661 cctagataca ttaccctgtt atcccagatg acataccctg ttatccctag atgacattac
      721 cctgttatcc cagataaact caatgatgat gatgatgatg gtcgagactc agcggccgcg
      781 gtgccagggc gtgcccttgg gctccccggg cgcgacgtga cgctgctgac ccctttcttt
      841 cccttctgca gggttccgga ggagagggca gaggaagtct gctaacatgc ggtgacgtcg
      901 aggagaatcc tggcccagaa ataggtactg gatttccctt tgacccccac tatgtggagg
      961 tgcttgggga acgaatgcac tatgttgacg tgggtcccag agatgggacc cccgtgttgt
     1021 ttttgcatgg aaatccaact agttcctatg tgtggcgaaa tataattcca catgtcgcac
     1081 ctacccaccg ctgtattgcc cccgacctca ttggtatggg aaaatctgat aagccagatt
     1141 tgggatattt ctttgatgac catgtgaggt ttatggacgc cttcattgaa gccttggggc
     1201 ttgaggaagt agtgctcgta attcacgatt gggggagcgc tctgggtttt cactgggcca
     1261 agaggaaccc agaaagggtt aagggtatcg catttatgga gttcatccgc ccaatcccaa
     1321 cttgggatga atggcctgag tttgcccgcg agacattcca ggcctttagg actaccgatg
     1381 ttggacggaa gttgattatc gatcagaacg tgtttataga gggaactttg ccaatggggg
     1441 tggtaaggcc cctgacagaa gtggaaatgg atcattatag ggagcccttc ttgaatcctg
     1501 tagacaggga acccctgtgg agattcccca acgaattgcc cattgcaggt gagcctgcca
     1561 atatcgtagc actcgtcgag gaatacatgg attggttgca tcaaagtccc gtcccaaaac
     1621 ttttgttctg gggaacccct ggtgttctca ttccaccagc agaggcagct cggttggcaa
     1681 aatcacttcc aaattgcaaa gcagtagata tcgggcctgg cctgaacctg ttgcaggagg
     1741 acaacccaga tttgataggc tcagaaatag cccgatggct gtcaactttg gagattagcg
     1801 gtgggtctgg tggcagtgga gggggatccg ggccagtacc caaaaagcgg gggatgaaaa
     1861 agcccgaact tacagctacc agtgtggaga aatttcttat tgaaaaattc gacagcgtat
     1921 cagatctgat gcaacttagc gaaggagagg aaagcagagc ttttagcttt gacgtgggcg
     1981 gacgagggta tgtactgcgc gtcaatagtt gtgcagatgg cttttacaag gatcggtacg
     2041 tctaccggca ctttgcatca gctgctttgc ctatccctga agtccttgac ataggagagt
     2101 tcagtgagag tctcacttac tgcatttcac gcagagcaca aggtgtgaca ctccaagacc
     2161 tccctgaaac cgagttgccc gccgttcttc aaccagtcgc cgaggcaatg gatgctatag
     2221 cagcagcaga tcttagccag accagcggct tcggtccatt cggcccacag ggaatcggtc
     2281 agtacacaac ttggcgagat tttatttgcg ctatagcaga cccccacgtc tatcactggc
     2341 agacagtaat ggacgacact gtgtcagcct ctgttgctca agctttggat gagcttatgc
     2401 tgtgggctga ggattgtcca gaagttcgcc acctcgtgca tgctgatttc ggttctaata
     2461 atgtacttac agataatggc aggataactg cagtcataga ctggagcgaa gcaatgtttg
     2521 gagactccca gtatgaggtc gcaaatattt ttttttggag gccatggctc gcctgtatgg
     2581 aacaacagac tcgatacttt gaacggcgcc accctgagct tgctggttct ccacggctcc
     2641 gggcctacat gctgaggata gggctcgatc aattgtacca atcattggtg gacggcaatt
     2701 ttgatgacgc tgcttgggct caaggtagat gtgacgctat cgtccggtcc ggtgcaggga
     2761 ccgtaggacg gactcagata gctagacgca gtgccgctgt atggactgat ggttgtgttg
     2821 aagtgcttgc cgactccggc aaccggcggc cttccacacg gccccgggct aaagaagtaa
     2881 gtcgactcgt tcgccccaac tggggtaacc tttgagttct ctcagttggg ggtaatcagc
     2941 atcatgatgt ggtaccacat catgatgctg attataagaa tgcggccgcc acactctagt
     3001 ggatctcgag ttaataattc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg
     3061 cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag
     3121 ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag
     3181 ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca
     3241 ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgctcgcct tgagcctggc
     3301 gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag
     3361 accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg
     3421 gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt
     3481 ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag
     3541 ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt
     3601 ggccagccac gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc
     3661 ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga
     3721 gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg
     3781 agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg
     3841 atcagagctt gatcccctgc gccatcagat ccttggcggc gagaaagcca tccagtttac
     3901 tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc
     3961 tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt
     4021 tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg
     4081 tcagcaccgt ttctgcggac tggctttcta cgtgctcgag gggggccaaa cggtctccag
     4141 cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa atcagaacgc
     4201 agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac
     4261 cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat
     4321 gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     4381 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
     4441 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata
     4501 aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct
     4561 acaaactctt ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaccaaaa
     4621 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     4681 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     4741 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     4801 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     4861 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     4921 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     4981 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5041 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5101 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     5161 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     5221 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     5281 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     5341 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     5401 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     5461 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc
     5521 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     5581 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5641 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5701 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag cagatcaatt cgcgcgcgaa
     5761 ggcgaagcgg catgcataat gtgcctgtca aatggacgaa gcagggattc tgcaaaccct
     5821 atgctactcc gtcaagccgt caattgtctg attcgttacc aattatgaca acttgacggc
     5881 tacatcattc actttttctt cacaaccggc acggaactcg ctcgggctgg ccccggtgca
     5941 ttttttaaat acccgcgaga aatagagttg atcgtcaaaa ccaacattgc gaccgacggt
     6001 ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc tggctgatac gttggtcctc
     6061 gcgccagctt aagacgctaa tccctaactg ctggcggaaa agatgtgaca gacgcgacgg
     6121 cgacaagcaa acatgctgtg cgacgctggc gat
//
