LOCUS       sequence-414896-        7253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HDR Universal Surrogate Reporter with a sgRNA and consistently 
            expressed eGFP but without Cas9. The selective PuroR reporter gene 
            was designed to be repaired only by the sgRNA/Cas9-triggered HDR..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7253)
  AUTHORS   Yan N, Sun Y, Fang Y, Deng J, Mu L, Xu K, Mymryk JS, Zhang Z
  TITLE     A Universal Surrogate Reporter for Efficient Enrichment of 
            CRISPR/Cas9-Mediated Homology-Directed Repair in Mammalian Cells.
  JOURNAL   Mol Ther Nucleic Acids. 2020 Mar 6;19:775-789. doi: 
            10.1016/j.omtn.2019.12.021. Epub 2019 Dec 24.
  PUBMED    31955009
REFERENCE   2  (bases 1 to 7253)
  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: "Mol Ther 
            Nucleic Acids."; date: "2020-03-6"; volume: "19:775-789. doi"; 
            pages: " 10.1016/j.omtn.2019.12.021. Epub 2019 Dec 24"
COMMENT     Sequence Label: pHDR-USR-eGFP
FEATURES             Location/Qualifiers
     source          1..7253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     169..188
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        267..342
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        383..762
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        763..966
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     917..937
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1568..1621
                     /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             join(1622..1624,1625..2338)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1622..1624,1625..2335)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1664..1685)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1925..1944)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2272..2293
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2374..2391)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2380..2604
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2658..2677
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2658..2676
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2691..3284
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     3295..3311
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3296..3312
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   3595..3629
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4120..4136)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4120..4136)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4133..4155)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4144..4160
                     /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(4168..4174,4175..4192,4193..4198))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4213..4234
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4351..4368)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4522..5110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4602..4621)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5281..6072,6073..6141))
                     /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     5904..5923
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6142..6246)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6272..6727)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6409..6430)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6621..6640
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6854..6876
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6868..6885
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6869..6885
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   7015..7077
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 gaattctttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
       61 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
      121 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
      181 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
      241 aacaccgttc tccgaacgtg tcacgtgttt tagagctaga aatagcaagt taaaataagg
      301 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttgc ggccgccgat
      361 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
      421 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      481 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      541 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      601 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      661 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggacttccct
      721 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      781 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      841 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      901 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      961 agagctctct ggctaactag agaacccact gcttactggc ttatcgaaat tggatccatg
     1021 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     1081 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     1141 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     1201 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggagttc tccgaacgtg
     1261 tcacgtggga ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac
     1321 cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg
     1381 agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa
     1441 cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg
     1501 accgcgcacc tggtgcatga cccgcaagcc cggtgccgct agcaccatgg aggtcgacgg
     1561 cagtggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc
     1621 agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1681 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     1741 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     1801 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     1861 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     1921 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1981 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     2041 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     2101 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2161 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2221 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2281 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaact
     2341 cgagggatca attctctagg gctcgctgat cagcctcgac tgtgccttct agttgccagc
     2401 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     2461 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     2521 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     2581 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgatcc
     2641 tctagagtcg aagatcttaa tacgactcac tatagggcga attgggtacc accgagtaca
     2701 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
     2761 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
     2821 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     2881 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     2941 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     3001 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     3061 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     3121 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     3181 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     3241 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgccgggccc cccctcgagg
     3301 tcgacggtat cgataagctt gatatctata acaagaaaat atatatataa taagttatca
     3361 cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg
     3421 taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca
     3481 cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa
     3541 tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg
     3601 tcaattttac gcagactatc tttctagggt taatctagct gcatcaggat catatcgtcg
     3661 ggtctttttt ccggctcagt catcgcccaa gctggcgcta tctgggcatc ggggaggaag
     3721 aagcccgtgc cttttcccgc gaggttgaag cggcatggaa agagtttgcc gaggatgact
     3781 gctgctgcat tgacgttgag cgaaaacgca cgtttaccat gatgattcgg gaaggtgtgg
     3841 ccatgcacgc ctttaacggt gaactgttcg ttcaggccac ctgggatacc agttcgtcgc
     3901 ggcttttccg gacacagttc cggatggtca gcccgaagcg catcagcaac ccgaacaata
     3961 ccggcgacag ccggaactgc cgtgccggtg tgcagattaa tgacagcggt gcggcgctgg
     4021 gatattacgt cagcgaggac gggtatcctg gctggatgcc gcagaaatgg acatggatac
     4081 cccgtgagtt acccggcggg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4141 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4201 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4261 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4321 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4381 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4441 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4501 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4561 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4621 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4681 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4741 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4801 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4861 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4921 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     4981 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5041 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5101 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5161 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5221 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5281 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5341 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5401 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5461 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5521 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5581 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5641 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5701 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5761 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5821 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5881 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     5941 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6001 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6061 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6121 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6181 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6241 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     6301 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     6361 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     6421 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     6481 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     6541 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     6601 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     6661 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     6721 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     6781 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
     6841 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcctcgttc
     6901 attcacgttt ttgaacccgt ggaggacggg cagactcgcg gtgcaaatgt gttttacagc
     6961 gtgatggagc agatgaagat gctcgacacg ctgcagaaca cgcagctaga ttaaccctag
     7021 aaagataatc atattgtgac gtacgttaaa gataatcatg tgtaaaattg acgcatgtgt
     7081 tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa gttttattat
     7141 atttacactt acatactaat aataaattca acaaacaatt tatttatgtt tatttattta
     7201 ttaaaaaaaa caaaaactca aaatttcttc tataaagtaa caaaactttt atc
//
