LOCUS       sequence-189981-        6268 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HMEJ donor for fusing a p2A-mCherry reporter to mouse Nanog. AAV 
            backbone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6268)
  AUTHORS   Yao X, Wang X, Hu X, Liu Z, Liu J, Zhou H, Shen X, Wei Y, Huang Z, 
            Ying W, Wang Y, Nie YH, Zhang CC, Li S, Cheng L, Wang Q, Wu Y, Huang
            P, Sun Q, Shi L, Yang H
  TITLE     Homology-mediated end joining-based targeted integration using 
            CRISPR/Cas9.
  JOURNAL   Cell Res. 2017 May 19. doi: 10.1038/cr.2017.76.
  PUBMED    28524166
REFERENCE   2  (bases 1 to 6268)
  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: "Cell Res. 
            2017 May 19. doi: 10.1038/cr.2017.76."
COMMENT     Sequence Label: AAV_Nanog HMEJ donor
FEATURES             Location/Qualifiers
     source          1..6268
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     134..153
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     387..404
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    521..542
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(557..562,563..580,581..587)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    595..611
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     600..622
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     619..635
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     619..635
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1498..1554
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1555..2265
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1700..1718)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1968..1987
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3115..3134)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3116..3134)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3141..3158)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3141..3157)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3150..3172)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3298..3726
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3385..3404)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3595..3616
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             4070..4864
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4124..4143)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4734..4753
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(4882..4950,4951..5742)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5100..5119)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5913..6268,1..233)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
       61 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
      121 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
      181 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
      241 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
      301 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
      361 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
      421 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
      481 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
      541 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
      601 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cttggtaccc
      661 gtaagtctca tatttcacct ggccccctcc atcttaccat cttattttat gtgtctgtgc
      721 cgtgtgtgtg tgtgtgtgtg tgtgtgtggc tgacctatgg ctgttgggta tggatcccag
      781 atttcctgta agagcagtca gtggctctgc ctttgcttcc cagtgtcatc ctgatccatc
      841 tatgtccatt agtcctgtcc ctttcccatc cctcttcctc cacgcactct ttcttgtgat
      901 ctttggttct ttgaggcagg ctctctctat gtagccctgg cagtcagaga tctgcctgcc
      961 tagtctcagg agtgctgggg ttaacggcct gtgcggccat gctaatcttt tcagtatttc
     1021 tgcagtttac atctttagag tccccttttc actcattgca ttaatcacct ctttcagaag
     1081 ggctcagcac cagtggagta tcccagcatc cattgcagct atccccaggg ctatctggtg
     1141 aacgcatctg gaagcctttc catgtggggc agccagactt ggaccaaccc aacttggagc
     1201 agccagacct ggaccaaccc aacttggaac aaccagacct ggaccaaccc aacttggagc
     1261 agccaggcct ggaccgctca gtcctggaac ggccagcctt ggaatgctgc tccgctccat
     1321 aacttcgggg aggactttct gcagccttac gtacagttgc agcaaaactt ctctgccagt
     1381 gatttggagg tgaatttgga agccactagg gaaagccatg cgcattttag caccccacaa
     1441 gccttggaat tattcctgaa ctactctgtg actccaccag gtgaaatatt aattaacgcc
     1501 actaacttct ccctgttgaa acaagcaggg gatgtcgaag agaatcccgg gccaatggtg
     1561 agcaagggcg aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac
     1621 atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc
     1681 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc
     1741 tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc
     1801 gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     1861 aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag
     1921 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     1981 aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg
     2041 aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc
     2101 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc
     2161 aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc
     2221 gagggccgcc actccaccgg cggcatggac gagctgtaca agtaaggcgc gccgaaatat
     2281 gagacttacg caacatctgg gcttaaagtc agggcaaagc caggttcctt ccttcttcca
     2341 aatattttca tatttttttt aaagatttat ttattcatta tatgtaagta cactgtagct
     2401 gtcttcagac actccagaag agggcgtcag atcttgttac gtatggttgt gagccaccat
     2461 gtggttgctg ggatttgaac tcctgacctt cggaagagca gtcgggtgct cttatccact
     2521 gagccatctc accagcccct ggtttatttt tttaattatt atttgctttt tgtttatcaa
     2581 gacagggttt ctctgcatag ctctaattgt ctttgaacta gctctgcaga ccagcctggc
     2641 cttgaactca gagatctgcc cacttatctt tgcctcctga atgctgggac caaaggtggc
     2701 ataccaccac acctggcata tatattgttt atttctattt ctatttttat tggtgccaga
     2761 gcaaacctag gacttagaac atgctgggca ccaactcaac ttctgagctc tatttacaac
     2821 ttggtgtgtt agtgtatttg tcttagttct gaatttgtcc tttttttagt gttaactcta
     2881 ggctttggag acagtgaggt gcatatactc tctccttccc aagaataagt gcttgaacac
     2941 ccttacccac gcccacccac ccatgctagt cttttttctt agaagcgtgg gtcttggtat
     3001 acactgtgtc attttgaggg gtgaggttta aaagtatata caaagtataa cgatatggtg
     3061 gctactctcg aggccaggtg aaatatgaga cttacgtcta gagggcccaa ttcgccctat
     3121 agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     3181 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     3241 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggacg
     3301 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     3361 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     3421 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     3481 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     3541 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     3601 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     3661 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     3721 cgaattttaa caaaattcag ggcgcaaggg ctgctaaagg aagcggaaca cgtagaaagc
     3781 cagtccgcag aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag
     3841 ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct
     3901 agactgggcg gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg
     3961 taaggttggg aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg
     4021 gcgcagggga tcaagatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4081 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4141 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4201 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     4261 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4321 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4381 atcccacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4441 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4501 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4561 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     4621 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4681 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4741 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4801 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4861 ctgaattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     4921 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     4981 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5041 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5101 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5161 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5221 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5281 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5341 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5401 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     5461 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     5521 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     5581 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     5641 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     5701 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     5761 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     5821 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     5881 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     5941 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6001 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6061 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6121 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6181 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6241 tcgtgcacac agcccagctt ggagcgaa
//
