LOCUS       sequence-413352-        5550 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Parent vector for producing universal minicircle donors for 
            mNeonGreen C-terminal knock-in.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5550)
  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 5550)
  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: pYS16
FEATURES             Location/Qualifiers
     source          1..5550
                     /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             1632..1685
                     /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             complement(2415..3209)
                     /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(2415..3209)
                     /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(2526..2545)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3136..3155
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(3573..3590)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3743..3829
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3921..3948
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4082..4670
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4571..4590
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4824..4841
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4856..4996
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4922..4941)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5082..5104
                     /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 ggcgcgacta gtgggccagt acccaaaaag cggggggtct ggtggcagtg
      901 gagggggatc catggttagt aaaggtgagg aggacaacat ggcaagtctc cccgctactc
      961 acgaacttca tatcttcggc tccataaacg gggttgactt tgatatggta gggcagggaa
     1021 cagggaatcc taatgacggc tatgaagagc tcaatttgaa gtcaaccaag ggtgatctcc
     1081 agttctcacc ctggattctg gtcccacata taggttacgg attccaccag taccttcctt
     1141 accctgatgg gatgagtcca tttcaagctg ccatggtaga tggtagcggg taccaggtcc
     1201 atcgcactat gcagtttgaa gacggtgctt cattgactgt taattaccgc tacacatacg
     1261 aaggctctca cattaaaggc gaagcccagg taaaaggcac aggatttccc gctgacggac
     1321 ctgttatgac aaattccctg accgcagcag actggtgcag gagtaaaaag acttatccca
     1381 atgataagac aattatcagt acattcaagt ggagctatac taccggcaac ggcaaaagat
     1441 atcgaagtac agcccggaca acttatacat ttgctaaacc aatggccgct aattatctca
     1501 aaaatcaacc catgtacgtc ttcagaaaaa cagagctgaa acacagtaag acagagctta
     1561 attttaaaga atggcaaaag gccttcaccg atgttatggg catggatgag ctctacaaag
     1621 gttccggagg agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg
     1681 gcccaatgac tgaatacaag cctactgtca ggttggctac aagagacgac gttcctagag
     1741 ccgtgagaac tctggctgca gccttcgccg actaccccgc cacgagacac accgttgacc
     1801 cagatcggca tattgagaga gtgactgaac tgcaggagct gtttcttaca agagttggcc
     1861 tcgacatagg caaggtgtgg gtggcggacg acggcgccgc cgtggccgtc tggaccactc
     1921 ccgaatcagt tgaggctggc gccgtattcg ctgagatcgg cccgagaatg gctgagctca
     1981 gcgggagtag gctcgcggca cagcagcaaa tggagggact gctggcacca cacaggccca
     2041 aagaacccgc ctggttcctg gcaaccgtcg gtgtatctcc cgatcatcag gggaaaggtc
     2101 tgggctctgc cgtagtgctc cctggcgtgg aggcagctga gagagcagga gtacctgcct
     2161 tcttggagac ctccgctcca aggaatcttc ccttctatga acggttgggc ttcaccgtga
     2221 cagccgacgt ggaagtcccc gaaggccccc gcacttggtg catgacgagg aagcctggag
     2281 cggggcccgc cccaactggg gtaacctttg agttctctca gttgggggta atcagcatca
     2341 tgatgtggta ccacatcatg atgctgatta taagaatgcg gccgccacac tctagtggat
     2401 ctcgagttaa taattcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa
     2461 tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct
     2521 tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg
     2581 ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca
     2641 tcgccatggg tcacgacgag atcctcgccg tcgggcatgc tcgccttgag cctggcgaac
     2701 agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg
     2761 gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag
     2821 gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg
     2881 gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag
     2941 tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc
     3001 agccacgata gccgcgctgc ctcgtcttgc agttcattca gggcaccgga caggtcggtc
     3061 ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag
     3121 ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa
     3181 cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca
     3241 gagcttgatc ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg
     3301 cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc
     3361 cataaaaccg cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc
     3421 tttgcgcttg cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag
     3481 caccgtttct gcggactggc tttctacgcg ctcgaggggg ggccaaacgg tctccagctt
     3541 ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc agaacgcaga
     3601 agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc
     3661 atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg
     3721 agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     3781 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc
     3841 ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac
     3901 tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca
     3961 aactcttttg tttatttttc taaatacatt caaatatgta tccgctcatg accaaaatcc
     4021 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4081 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4141 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4201 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4261 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4321 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4381 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4441 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4501 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     4561 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4621 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4681 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4741 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4801 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     4861 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca
     4921 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4981 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5041 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5101 gaggttttca ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc gcgcgaaggc
     5161 gaagcggcat gcataatgtg cctgtcaaat ggacgaagca gggattctgc aaaccctatg
     5221 ctactccgtc aagccgtcaa ttgtctgatt cgttaccaat tatgacaact tgacggctac
     5281 atcattcact ttttcttcac aaccggcacg gaactcgctc gggctggccc cggtgcattt
     5341 tttaaatacc cgcgagaaat agagttgatc gtcaaaacca acattgcgac cgacggtggc
     5401 gataggcatc cgggtggtgc tcaaaagcag cttcgcctgg ctgatacgtt ggtcctcgcg
     5461 ccagcttaag acgctaatcc ctaactgctg gcggaaaaga tgtgacagac gcgacggcga
     5521 caagcaaaca tgctgtgcga cgctggcgat
//
