LOCUS       sequence-218254-        6770 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter for canonical-NHEJ. Reporter for end joining between two 
            double-strand breaks, induced with the 7a and 7b sgRNAs with CAS9. 
            EJ between the distal ends w/o indel mutations restores GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6770)
  AUTHORS   Bhargava R, Sandhu M, Muk S, Lee G, Vaidehi N, Stark JM
  TITLE     C-NHEJ without indels is robust and requires synergistic function of
            distinct XLF domains.
  JOURNAL   Nat Commun. 2018 Jun 27;9(1):2484. doi: 10.1038/s41467-018-04867-5.
  PUBMED    29950655
REFERENCE   2  (bases 1 to 6770)
  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: "Nat 
            Commun."; date: "2018-06-27"; volume: "9(1):2484. doi"; pages: " 
            10.1038/s41467-018-04867-5"
COMMENT     Sequence Label: pCMV6-AC EJ7-GFP
FEATURES             Location/Qualifiers
     source          1..6770
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     126..148
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     140..157
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     852..872
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     927..946
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        927..945
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      960..969
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1011..1032)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1318..1337)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1665..1686
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1863..1916
                     /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"
     primer_bind     complement(1950..1966)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1977..2599
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2176..2195)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2623..2643)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2628..2957
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2808..2943
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3024..3818
                     /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(3078..3097)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3688..3707
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3992..4113
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4029..4048)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4083..4102
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4162..4178)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4175..4197)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4186..4202
                     /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)."
     promoter        complement(join(4210..4216,4217..4234,4235..4240))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4255..4276
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4393..4410)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4564..5152)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4644..4663)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5323..6114,6115..6183))
                     /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     5946..5965
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6184..6288)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6315..6743
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6402..6421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6612..6633
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa
      181 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta
      241 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      301 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
      361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      541 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      841 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      901 tcgtttagtg aaccgtcaga attttgtaat acgactcact atagggcggc cgggaattcg
      961 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1021 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1081 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1141 ccaccctcgt gaccaccctg acctacggct aaggaagata tctagggata acagggtaat
     1201 cccgggggcc tattcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1261 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1321 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1381 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1441 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1501 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     1561 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1621 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1681 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aggccggcca
     1741 gccacggctt cccccctgag gtggccgctc aggacgatgg caccctgccc atgagctgcg
     1801 cccaggagag cggcatggac aggcaccccg ccgcttgcgc cagcgctagg atcaacgtgg
     1861 gtgagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccg ggccctgtgg
     1921 taccctcgag gtttaaacgg ccggccgcgg tcatagctgt ttcctgaaca gatcccgggt
     1981 ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc actccagtgc
     2041 ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag gtgtccttct
     2101 ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg aagacaacct
     2161 gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa tcttggctca
     2221 ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagttgttgg
     2281 gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag agacggggtt
     2341 tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac ccaccttggc
     2401 ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc cttctgattt
     2461 taaaataact ataccagcag gaggacgtcc agacacagca taggctacct ggccatgccc
     2521 aaccggtggg acatttgagt tgcttgcttg gcactgtcct ctcatgcgtt gggtccactc
     2581 agtagatgcc tgttgaattg ggtacgcggc cagcttggct gtggaatgtg tgtcagttag
     2641 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2701 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2761 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2821 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2881 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2941 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     3001 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     3061 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3121 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3181 tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga
     3241 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3301 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3361 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3421 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3481 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3541 ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3601 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3661 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3721 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3781 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     3841 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     3901 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3961 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     4021 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4081 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4141 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4201 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4261 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4321 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4381 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4441 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4501 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4561 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4621 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4681 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4741 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4801 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4861 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4921 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4981 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5041 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5101 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5161 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5221 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5281 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5341 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5401 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5461 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5521 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5581 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5641 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5701 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5761 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5821 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5881 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5941 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6001 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6061 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6121 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6181 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6241 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6301 aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     6361 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     6421 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     6481 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     6541 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     6601 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     6661 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     6721 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
//
