LOCUS       sequence-373233-        7120 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Dual-fluorescent surrogate reporter vector for in vitro functional 
            assay of programable DNA nuclease and enrichment of genetically 
            edited cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7120)
  AUTHORS   Zhou Y, Liu Y, Hussmann D, Brogger P, Al-Saaidi RA, Tan S, Lin L, 
            Petersen TS, Zhou GQ, Bross P, Aagaard L, Klein T, Ronn SG, Pedersen
            HD, Bolund L, Nielsen AL, Sorensen CB, Luo Y
  TITLE     Enhanced genome editing in mammalian cells with a modified 
            dual-fluorescent surrogate system.
  JOURNAL   Cell Mol Life Sci. 2016 Jan 11.
  PUBMED    26755436
REFERENCE   2  (bases 1 to 7120)
  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 Mol 
            Life Sci. 2016 Jan 11."
COMMENT     Sequence Label: P119 C-Check
FEATURES             Location/Qualifiers
     source          1..7120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             426..1217
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1310..1898
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1799..1818
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2052..2069
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2228..2255)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2347..2433)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2497..2513
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2574..3073
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2609..2632)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3006..3025
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      3097..3106
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3103..3105,3106..3108,3109..3621)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     complement(3148..3169)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(3599..3994)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWEVSRLSGCRAAA
                     RGRRRWGCSAGSGRRAARCRPRCCGGS"
     primer_bind     3859..3875
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3885..3904
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3885..3903
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3951..3971)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3951..3969)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3990..4006)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3990..4006)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4003..4025)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4014..4030
                     /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(4038..4044,4045..4062,4063..4068))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4083..4104
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     4698..4719
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4887..5008
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4924..4943)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4978..4997
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     enhancer        5021..5400
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5401..5604
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5555..5575
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      6163..6172
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6169..6867
                     /codon_start=1
                     /product="Anemonia sulcata red fluorescent protein"
                     /label=AsRed2
                     /note="mammalian codon-optimized"
                     /translation="MASLLKKTMPFRTTIEGTVNGHYFKCTGKGEGNPLEGTQEMKIEV
                     IEGGPLPFAFHILSTSCMYGSKAFIKYVSGIPDYFKQSLPEGFTWERTTTYEDGGFLTA
                     HQDTSLDGDCLVYKVKILGNNFPADGPVMQNKAGRWEPSTEIVYEVDGVLRGQSLMALE
                     CPGGRHLTCHLHTTYRSKKPASALKMPGFHFEDHRIEILEEVEKGKCYKQYEAAVGRYC
                     DAAPSKLGHN"
     polyA_signal    6890..7114
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gcaagcaagc gctcgaaacg gtgcagcggc tgttgccggt gctgtgccag gaccatggcc
       61 gagacgtcta gaccagccag gacagaaatg cctcgacttc gctgctaccc aaggttgccg
      121 ggtgacgcac accgtggaaa cggatgaagg cacgaaccca gtggacataa gcctgttcgg
      181 ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga accttgaccg
      241 aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac tgtttttttg
      301 gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt
      361 tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa acaaagttaa
      421 acattatgag ggaagcggtg atcgccgaag tatcgactca actatcagag gtagttggcg
      481 tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg
      541 atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc gtaaggcttg
      601 atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct tcccctggag
      661 agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac atcattccgt
      721 ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat gacattcttg
      781 caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg acaaaagcaa
      841 gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat ccggttcctg
      901 aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg ccgcccgact
      961 gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac agcgcagtaa
     1021 ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc ctgccggccc
     1081 agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa gaagatcgct
     1141 tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc gagatcacca
     1201 aggtagtcgg caaataaccc tcgagccacc catgaccaaa atcccttaac gtgagttacg
     1261 cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1321 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1381 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1441 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     1501 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     1561 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     1621 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     1681 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     1741 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     1801 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     1861 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     1921 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     1981 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2041 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     2101 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     2161 aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc tagccaggaa gagtttgtag
     2221 aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta
     2281 tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc
     2341 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
     2401 ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcgt
     2461 taacgctagc atggatgttt tcccagtcac gacgttgtaa aacgacggcc agtcttaagc
     2521 gtctccccct gaacctgacc ccggaccaag tggtggctat atggtcgagt accgggtagg
     2581 ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg
     2641 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc
     2701 ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt
     2761 cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac
     2821 tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag
     2881 cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc
     2941 cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg
     3001 cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc
     3061 tccgggcctt tcgacctcta gcgggatcca ccggtcgcca ccatggtgag caagggcgag
     3121 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     3181 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     3241 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     3301 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     3361 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     3421 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     3481 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     3541 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     3601 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     3661 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actgagtcgt gagacctccc
     3721 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
     3781 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
     3841 tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat
     3901 agggcgaatt gggtaccggg ccccccctcg aggtcctcca gcttttgttc cctttagtga
     3961 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     4021 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     4081 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttg gtctcaaccg
     4141 tccagcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     4201 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     4261 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     4321 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     4381 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     4441 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     4501 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     4561 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     4621 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     4681 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     4741 ggcatggacg agctgtacaa gtaaagcggc cgcgactcta gatcataatc agccatacca
     4801 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     4861 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     4921 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4981 gtttgtccaa actcatcaat gtatcttaag gcgtagtgtt gacattgatt attgactagt
     5041 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     5101 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     5161 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     5221 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     5281 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     5341 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     5401 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     5461 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     5521 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     5581 tgggaggtct atataagcag agctcgtcga cttcgaattt ttcgcaacgg gtttgccgcc
     5641 agaacacagg tacacatatt gaccaaatca gggtaatttt gcatttgtaa ttttaaaaaa
     5701 tgctttcttc ttttaatata cttttttgtt tatcttattt ctaatacttt ccctaatctc
     5761 tttctttcag ggcaataatg atacaatgta tcatgcctct ttgcaccatt ctaaagaata
     5821 acagtgataa tttctgggtt aaggcaatag ccgattagtt cgtacgggcc ggccatgcat
     5881 cttttggaga atatttctgc atataaatat ttctgcatat aaattgtaac tgatgtaaga
     5941 ggtttcatat tgctaatagc agctacaatc cagctaccat tctgctttta ttttatggtt
     6001 gggataaggc tggattattc tgagtccaag ctaggccctt ttgctaatca tgttcatacc
     6061 tcttatcttc ctcccacagc tcctgggcaa cgtgctggtc tgtgtgctgg cccatcactt
     6121 tggcaaagca cgtgagatct gaattctgac acacccccgg gcgccaccat ggcctctttg
     6181 ctgaaaaaga ccatgccctt caggaccacc atcgagggca ccgtgaacgg ccactacttc
     6241 aagtgcaccg gcaagggcga gggcaacccc ctggagggca cccaggagat gaagatcgag
     6301 gtgatcgagg gcggccccct gcccttcgcc ttccacatcc tgtccacctc ctgcatgtac
     6361 ggctccaagg ccttcatcaa gtacgtgtcc ggcatccccg actacttcaa gcagtccctc
     6421 cccgagggct tcacctggga gcgcaccacc acctacgagg acggcggctt cctgaccgcc
     6481 caccaggaca cctccctgga cggcgactgc ctggtgtaca aggtgaagat cctgggcaac
     6541 aacttccccg ccgacggccc cgtgatgcag aacaaggccg gccgctggga gccctccacc
     6601 gagatcgtgt acgaggtgga cggcgtgctg cgcggccagt ccctgatggc cctggagtgc
     6661 cccggcggtc gccacctgac ctgccacctg cacaccacct accgctccaa gaagcccgcc
     6721 tccgccctga agatgcccgg cttccacttc gaggaccacc gcatcgagat cctggaggag
     6781 gtggagaagg gcaagtgcta caagcagtac gaggccgccg tgggccgcta ctgcgacgcc
     6841 gccccctcca agctgggcca caactgaagc ttctagaccc gggttcgaac tgtgccttct
     6901 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     6961 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     7021 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     7081 agcaggcatg ctggggatgc ggtgggctct atggctggcg
//
