LOCUS       sequence-409950-        5602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Parent vector for producing universal minicircle donors for 
            mNeonGreen N-terminal knock-in.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5602)
  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 5602)
  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: pYS17
FEATURES             Location/Qualifiers
     source          1..5602
                     /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             910..963
                     /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(2467..3261)
                     /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(2467..3261)
                     /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(2578..2597)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3188..3207
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(3625..3642)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3795..3881
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3973..4000
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      4134..4722
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4623..4642
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4876..4893
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4908..5048
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4974..4993)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5134..5156
                     /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 gtgtgacgct gctgacccct ttctttccct tctgcagggt
      901 tccggaggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc
      961 ccaatggtta gtaaaggtga ggaggacaac atggcaagtc tccccgctac tcacgaactt
     1021 catatcttcg gctccataaa cggggttgac tttgatatgg tagggcaggg aacagggaat
     1081 cctaatgacg gctatgaaga gctcaatttg aagtcaacca agggtgatct ccagttctca
     1141 ccctggattc tggtcccaca tataggttac ggattccacc agtaccttcc ttaccctgat
     1201 gggatgagtc catttcaagc tgccatggta gatggtagcg ggtaccaggt ccatcgcact
     1261 atgcagtttg aagacggtgc ttcattgact gttaattacc gctacacata cgaaggctct
     1321 cacattaaag gcgaagccca ggtaaaaggc acaggatttc ccgctgacgg acctgttatg
     1381 acaaattccc tgaccgcagc agactggtgc aggagtaaaa agacttatcc caatgataag
     1441 acaattatca gtacattcaa gtggagctat actaccggca acggcaaaag atatcgaagt
     1501 acagcccgga caacttatac atttgctaaa ccaatggccg ctaattatct caaaaatcaa
     1561 cccatgtacg tcttcagaaa aacagagctg aaacacagta agacagagct taattttaaa
     1621 gaatggcaaa aggccttcac cgatgttatg ggcatggatg agctctacaa agggtctggt
     1681 ggcagtggag ggggatccgg gccagtaccc aaaaagcggg ggatgactga atacaagcct
     1741 actgtcaggt tggctacaag agacgacgtt cctagagccg tgagaactct ggctgcagcc
     1801 ttcgccgact accccgccac gagacacacc gttgacccag atcggcatat tgagagagtg
     1861 actgaactgc aggagctgtt tcttacaaga gttggcctcg acataggcaa ggtgtgggtg
     1921 gcggacgacg gcgccgccgt ggccgtctgg accactcccg aatcagttga ggctggcgcc
     1981 gtattcgctg agatcggccc gagaatggct gagctcagcg ggagtaggct cgcggcacag
     2041 cagcaaatgg agggactgct ggcaccacac aggcccaaag aacccgcctg gttcctggca
     2101 accgtcggtg tatctcccga tcatcagggg aaaggtctgg gctctgccgt agtgctccct
     2161 ggcgtggagg cagctgagag agcaggagta cctgccttct tggagacctc cgctccaagg
     2221 aatcttccct tctatgaacg gttgggcttc accgtgacag ccgacgtgga agtccccgaa
     2281 ggcccccgca cttggtgcat gacgaggaag cctggagcgg taagtcgact cgttgggccc
     2341 gccccaactg gggtaacctt tgagttctct cagttggggg taatcagcat catgatgtgg
     2401 taccacatca tgatgctgat tataagaatg cggccgccac actctagtgg atctcgagtt
     2461 aataattcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc
     2521 ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat
     2581 atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc
     2641 gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg
     2701 ggtcacgacg agatcctcgc cgtcgggcat gctcgccttg agcctggcga acagttcggc
     2761 tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat
     2821 ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg
     2881 atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc
     2941 aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc
     3001 cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga
     3061 tagccgcgct gcctcgtctt gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa
     3121 aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt
     3181 ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg
     3241 caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cagagcttga
     3301 tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt tgcagggctt
     3361 cccaacctta ccagagggcg ccccagctgg caattccggt tcgcttgctg tccataaaac
     3421 cgcccagtct agctatcgcc atgtaagccc actgcaagct acctgctttc tctttgcgct
     3481 tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc agcaccgttt
     3541 ctgcggactg gctttctacg cgctcgaggg ggggccaaac ggtctccagc ttggctgttt
     3601 tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct
     3661 gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa
     3721 ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg cgagagtagg
     3781 gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     3841 tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
     3901 acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa actgccaggc
     3961 atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta caaactcttt
     4021 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaccaaaat cccttaacgt
     4081 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4141 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4201 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4261 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     4321 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4381 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4441 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4501 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4561 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4621 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4681 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4741 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4801 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4861 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4921 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     4981 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     5041 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     5101 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     5161 caccgtcatc accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc
     5221 atgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta tgctactccg
     5281 tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct acatcattca
     5341 ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat tttttaaata
     5401 cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg gcgataggca
     5461 tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg cgccagctta
     5521 agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa
     5581 catgctgtgc gacgctggcg at
//
