LOCUS       sequence-400073-        6196 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 6196)
  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 6196)
  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..6196
                     /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             906..959
                     /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             960..1844
                     /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             1896..2918
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(3061..3855)
                     /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(3061..3855)
                     /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(3172..3191)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3782..3801
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4219..4236)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4389..4475
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4567..4594
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4728..5316
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5217..5236
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5470..5487
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5502..5642
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5568..5587)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5728..5750
                     /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 cagggttccg
      901 gaggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccag
      961 aaataggtac tggatttccc tttgaccccc actatgtgga ggtgcttggg gaacgaatgc
     1021 actatgttga cgtgggtccc agagatggga cccccgtgtt gtttttgcat ggaaatccaa
     1081 ctagttccta tgtgtggcga aatataattc cacatgtcgc acctacccac cgctgtattg
     1141 cccccgacct cattggtatg ggaaaatctg ataagccaga tttgggatat ttctttgatg
     1201 accatgtgag gtttatggac gccttcattg aagccttggg gcttgaggaa gtagtgctcg
     1261 taattcacga ttgggggagc gctctgggtt ttcactgggc caagaggaac ccagaaaggg
     1321 ttaagggtat cgcatttatg gagttcatcc gcccaatccc aacttgggat gaatggcctg
     1381 agtttgcccg cgagacattc caggccttta ggactaccga tgttggacgg aagttgatta
     1441 tcgatcagaa cgtgtttata gagggaactt tgccaatggg ggtggtaagg cccctgacag
     1501 aagtggaaat ggatcattat agggagccct tcttgaatcc tgtagacagg gaacccctgt
     1561 ggagattccc caacgaattg cccattgcag gtgagcctgc caatatcgta gcactcgtcg
     1621 aggaatacat ggattggttg catcaaagtc ccgtcccaaa acttttgttc tggggaaccc
     1681 ctggtgttct cattccacca gcagaggcag ctcggttggc aaaatcactt ccaaattgca
     1741 aagcagtaga tatcgggcct ggcctgaacc tgttgcagga ggacaaccca gatttgatag
     1801 gctcagaaat agcccgatgg ctgtcaactt tggagattag cggtgggtct ggtggcagtg
     1861 gagggggatc cgggccagta cccaaaaagc gggggatgaa aaagcccgaa cttacagcta
     1921 ccagtgtgga gaaatttctt attgaaaaat tcgacagcgt atcagatctg atgcaactta
     1981 gcgaaggaga ggaaagcaga gcttttagct ttgacgtggg cggacgaggg tatgtactgc
     2041 gcgtcaatag ttgtgcagat ggcttttaca aggatcggta cgtctaccgg cactttgcat
     2101 cagctgcttt gcctatccct gaagtccttg acataggaga gttcagtgag agtctcactt
     2161 actgcatttc acgcagagca caaggtgtga cactccaaga cctccctgaa accgagttgc
     2221 ccgccgttct tcaaccagtc gccgaggcaa tggatgctat agcagcagca gatcttagcc
     2281 agaccagcgg cttcggtcca ttcggcccac agggaatcgg tcagtacaca acttggcgag
     2341 attttatttg cgctatagca gacccccacg tctatcactg gcagacagta atggacgaca
     2401 ctgtgtcagc ctctgttgct caagctttgg atgagcttat gctgtgggct gaggattgtc
     2461 cagaagttcg ccacctcgtg catgctgatt tcggttctaa taatgtactt acagataatg
     2521 gcaggataac tgcagtcata gactggagcg aagcaatgtt tggagactcc cagtatgagg
     2581 tcgcaaatat ttttttttgg aggccatggc tcgcctgtat ggaacaacag actcgatact
     2641 ttgaacggcg ccaccctgag cttgctggtt ctccacggct ccgggcctac atgctgagga
     2701 tagggctcga tcaattgtac caatcattgg tggacggcaa ttttgatgac gctgcttggg
     2761 ctcaaggtag atgtgacgct atcgtccggt ccggtgcagg gaccgtagga cggactcaga
     2821 tagctagacg cagtgccgct gtatggactg atggttgtgt tgaagtgctt gccgactccg
     2881 gcaaccggcg gccttccaca cggccccggg ctaaagaagt aagtcgactc gttcgcccca
     2941 actggggtaa cctttgagtt ctctcagttg ggggtaatca gcatcatgat gtggtaccac
     3001 atcatgatgc tgattataag aatgcggccg ccacactcta gtggatctcg agttaataat
     3061 tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat
     3121 accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
     3181 ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa
     3241 tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac
     3301 gacgagatcc tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc
     3361 gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt
     3421 acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
     3481 cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg
     3541 agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc
     3601 agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
     3661 cgctgcctcg tcttgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac
     3721 cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
     3781 tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc
     3841 atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagagc ttgatcccct
     3901 gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac
     3961 cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca
     4021 gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt
     4081 ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg
     4141 actggctttc tacgcgctcg agggggggcc aaacggtctc cagcttggct gttttggcgg
     4201 atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa
     4261 acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga
     4321 agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg
     4381 ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     4441 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     4501 cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa
     4561 ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttttgttta
     4621 tttttctaaa tacattcaaa tatgtatccg ctcatgacca aaatccctta acgtgagttt
     4681 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4741 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4801 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4861 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4921 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4981 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5041 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5101 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5161 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5221 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5281 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5341 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5401 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5461 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     5521 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     5581 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     5641 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5701 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5761 catcaccgaa acgcgcgagg cagcagatca attcgcgcgc gaaggcgaag cggcatgcat
     5821 aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac tccgtcaagc
     5881 cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca ttcacttttt
     5941 cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg
     6001 agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg
     6061 tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc
     6121 taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct
     6181 gtgcgacgct ggcgat
//
