LOCUS       sequence-97601-e        4781 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP reporter for CRISPR/CAS9 and gRNA_AAVS1-T2 activities.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4781)
  AUTHORS   Zhang JH, Pandey M, Kahler JF, Loshakov A, Harris B, Dagur PK, Mo Y,
            Simonds WF
  TITLE     Improving the specificity and efficacy of CRISPR/CAS9 and gRNA 
            through target specific DNA reporter.
  JOURNAL   J Biotechnol. 2014 Sep 2. pii: S0168-1656(14)00838-4. doi: 
            10.1016/j.jbiotec.2014.08.033.
  PUBMED    25193712
REFERENCE   2  (bases 1 to 4781)
  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: "J 
            Biotechnol. 2014 Sep 2. pii: S0168-1656(14)00838-4. doi: 
            10.1016/j.jbiotec.2014.08.033."
COMMENT     Sequence Label: psAAVS1-T2
FEATURES             Location/Qualifiers
     source          1..4781
                     /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      653..662
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(659..661,662..664,665..1375)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(659..661,662..664,665..1375)
                     /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(704..725)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(965..984)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1312..1333
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1389..1445
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    1569..1690
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1606..1625)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1660..1679
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1697..2152)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1834..1855)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2046..2065
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2179..2283
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2285..2642
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2285..2476
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2308..2328)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2493..2628
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2677..3471
                     /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             2677..3471
                     /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(2731..2750)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3341..3360
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3659..3678)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3703..3750
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4079..4667
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4568..4587
                     /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 tgtatgagac
      601 cggggccact agggacagga ttggggtctc tgtttcgcta gcgctaccgg tcgccaccat
      661 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      721 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
      781 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
      841 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
      901 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
      961 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1021 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     1081 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     1141 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     1201 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1261 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1321 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtccgg
     1381 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
     1441 gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac
     1501 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     1561 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     1621 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     1681 atgtatctta acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     1741 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     1801 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     1861 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     1921 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     1981 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     2041 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     2101 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     2161 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2221 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2281 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2341 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2401 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2461 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     2521 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     2581 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     2641 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     2701 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     2761 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     2821 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     2881 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     2941 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3001 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3061 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3121 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3181 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     3241 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3301 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3361 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3421 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     3481 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     3541 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     3601 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     3661 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     3721 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     3781 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     3841 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     3901 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     3961 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4021 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4081 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4141 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4201 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     4261 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4321 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4381 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4441 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4501 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4561 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4621 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4681 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4741 tatcccctga ttctgtggat aaccgtatta ccgccatgca t
//
