LOCUS       sequence-178920-        4658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NHEJ donor for fusing a p2A-mCherry reporter to mouse Actb..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4658)
  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 4658)
  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_Actb NHEJ donor
FEATURES             Location/Qualifiers
     source          1..4658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             270..326
                     /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             327..1037
                     /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(472..490)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     740..759
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1091..1110)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1092..1110)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1117..1134)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1117..1133)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1126..1148)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1274..1702
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1361..1380)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1571..1592
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             2046..2840
                     /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(2100..2119)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2710..2729
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(2858..2926,2927..3718)
                     /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(3076..3095)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3889..4477
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4378..4397
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4631..4648
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 gtaccagtcc gcctagaagc acttgcggcg ccactaactt ctccctgttg aaacaagcag
      301 gggatgtcga agagaatccc gggccaatgg tgagcaaggg cgaggaggat aacatggcca
      361 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt
      421 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
      481 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt
      541 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
      601 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
      661 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
      721 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct
      781 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc
      841 tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg
      901 tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg
      961 actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg
     1021 acgagctgta caagtaaagt ccgcctagaa gcacttgcgg ctcgagcatg catctagagg
     1081 gcccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg
     1141 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     1201 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     1261 gaatggcgaa tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     1321 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     1381 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     1441 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     1501 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     1561 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     1621 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1681 taacaaaaat ttaacgcgaa ttttaacaaa attcagggcg caagggctgc taaaggaagc
     1741 ggaacacgta gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact
     1801 gggctatctg gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc
     1861 ttacatggcg atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag
     1921 ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc
     1981 cgccaaggat ctgatggcgc aggggatcaa gatctgatca agagacagga tgaggatcgt
     2041 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     2101 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     2161 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     2221 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     2281 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     2341 ggcaggatct cctgtcatcc caccttgctc ctgccgagaa agtatccatc atggctgatg
     2401 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     2461 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     2521 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     2581 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     2641 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     2701 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     2761 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     2821 ttcttgacga gttcttctga attgaaaaag gaagagtatg agtattcaac atttccgtgt
     2881 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2941 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3001 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3061 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3121 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3181 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3241 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3301 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3361 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     3421 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3481 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3541 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     3601 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3661 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     3721 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     3781 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     3841 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3901 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3961 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4021 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4081 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4141 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4201 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4261 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4321 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4381 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4441 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4501 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4561 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4621 gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
