LOCUS       sequence-271055-        5810 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SSR2.0 has two target sites in opposite orientation for generate 
            only one cycle of repairing after I-SceI endonuclease expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5810)
  AUTHORS   Gomez-Cabello D, Jimeno S, Fernandez-Avila MJ, Huertas P
  TITLE     New tools to study DNA double-strand break repair pathway choice.
  JOURNAL   PLoS One. 2013 Oct 14;8(10):e77206. doi: 
            10.1371/journal.pone.0077206.
  PUBMED    24155929
REFERENCE   2  (bases 1 to 5810)
  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: "PLoS One.";
            date: "2013-10-14"; volume: "8(10):e77206. doi"; pages: " 
            10.1371/journal.pone.0077206"
COMMENT     Sequence Label: SeeSaw Reporter (SSR2.0)
FEATURES             Location/Qualifiers
     source          1..5810
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1045..1054
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1051..1053,1054..1056,1057..1767)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1051..1053,1054..1056,1057..1767)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1096..1117)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1357..1376)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1704..1725
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2598..2719
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2635..2654)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2689..2708
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2726..3181)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2863..2884)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3075..3094
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3208..3312
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3314..3671
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3314..3505
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3337..3357)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3522..3657
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3706..4500
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3706..4500
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3760..3779)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4370..4389
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4688..4707)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4732..4779
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5108..5696
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5597..5616
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag cggcctgctg aaggagagta tgcgcatcaa gatgtacatg
      661 gagggcaccg tgaacggcca ctacttcaag tgcgagggcg agggcgacgg caaccccttc
      721 gccggcaccc agagcatgag aatccacgtg accgagggcg cccccctgcc cttcgccttc
      781 gacatcctgg ccccctgctg cgagtacggc agcaggacct tcgtgcacca caccgccgag
      841 atccccgact tcttcaagca gagcttcccc gagggcttca cctgggagag aaccaccacc
      901 tacgaggacg gcggcatcct gaccgcccac caggacacca gcctggaggg caactgcctg
      961 atctacaagg tgaaagtgca cggcaccaac ttccccgccg acggccccgt gatgaagaac
     1021 aagagcggtc tagcgctacc ggtcgccacc atggtgagca agggcgagga gctgttcacc
     1081 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1141 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1201 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1261 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1321 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1381 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1441 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1501 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1561 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1621 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     1681 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1741 actctcggca tggacgagct gtacaagtcc ggactcagat ctcgagctca agcttcgaat
     1801 tctagggata acagggtaat aataattacc ctgttatccc tatgaattct gcagtcgaca
     1861 cccagagcat gagaatccac gtgaccgagg gcgcccccct gcccttcgcc ttcgacatcc
     1921 tggccccctg ctgcgagtac ggcagcagga ccttcgtgca ccacaccgcc gagatccccg
     1981 acttcttcaa gcagagcttc cccgagggct tcacctggga gagaaccacc acctacgagg
     2041 acggcggcat cctgaccgcc caccaggaca ccagcctgga gggcaactgc ctgatctaca
     2101 aggtgaaggt gcacggcacc aacttccccg ccgacggccc cgtgatgaag aacaagagcg
     2161 gcggctggaa gcccagcacc gaggtggtgt accccgagaa cggcgtgctg tgcggccgga
     2221 acgtgatggc cctgaaggtg ggcgaccggc acctgatctg ccaccactac accagctacc
     2281 ggagcaagaa ggccgtgcgc gccctgacca tgcccggctt ccacttcacc gacatccggc
     2341 tccagatgct gcggaagaag aaggacgagt acttcgagct gtacgaggcc agcgtggccc
     2401 ggtacagcga cctgcccgag aaggccaact ccggactcag atctcgagct caagcttcga
     2461 attctgcagg atccaccgga tctagataac tgatcataat cagccatacc acatttgtag
     2521 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     2581 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     2641 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     2701 aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     2761 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     2821 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     2881 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     2941 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     3001 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     3061 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     3121 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     3181 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3241 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3301 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     3361 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3421 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3481 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3541 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3601 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3661 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     3721 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3781 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3841 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     3901 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3961 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4021 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4081 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4141 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4201 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     4261 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4321 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4381 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4441 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4501 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4561 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4621 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     4681 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     4741 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     4801 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     4861 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     4921 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
     4981 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5041 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5101 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5161 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5221 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5281 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5341 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5401 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5461 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5521 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5581 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5641 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5701 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5761 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
