LOCUS       sequence-434404-        6599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter plasmid that encodes for the GFP fused to an N-terminal 
            flag-HA epitope. A stop codon is inserted between the epitope tag 
            and the gfp sequence to terminate GFP translation..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6599)
  AUTHORS   Nemudraia A, Nemudryi A, Wiedenheft B
  TITLE     Repair of CRISPR-guided RNA breaks enables site-specific RNA 
            excision in human cells.
  JOURNAL   Science. 2024 Apr 25:eadk5518. doi: 10.1126/science.adk5518.
  PUBMED    38662916
REFERENCE   2  (bases 1 to 6599)
  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: "Science. 
            2024 Apr 25:eadk5518. doi: 10.1126/science.adk5518."
COMMENT     Sequence Label: pAN007
FEATURES             Location/Qualifiers
     source          1..6599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             118..126
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             complement(133..159)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(175..198)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      198..207
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(290..493)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(319..339)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        494..797
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(972..1560)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1052..1071)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    1889..1936
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     1961..1980
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(2168..2962)
                     /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             complement(2168..2962)
                     /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(2279..2298)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2889..2908
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2997..3354)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3011..3146
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3163..3354
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3311..3331
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3356..3460)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3487..3942
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3574..3593)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3784..3805
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    3949..4070
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3960..3979)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4014..4033
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4194..5846)
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     primer_bind     5762..5780
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             complement(5853..5906)
                     /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"
ORIGIN
        1 catcttgttg gtcatgcggc cctgctcggg ggtgccctct ccgccgccca ccagctcgaa
       61 ctccacgccg ttcagggtgc cggtgatgcg gcactcgatc ttggcggcgg gggcagaacc
      121 tccttatcca ccagcgtaat ctggaacatc gtatgggtaa cctccaccgc cccctttatc
      181 gtcatcgtct ttgtagtcca tggtggcggt accgtcgact gcagaattcg aagcttgagc
      241 tcgagatctg agtccggtag cgctagcgga tctgacggtt cactaaacca gctctgctta
      301 tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac
      361 gacattttgg aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg
      421 ggtggagact tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc
      481 aaaaccgcat caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa
      541 agtcccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt
      601 caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc
      661 gtaaatactc cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat
      721 acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta
      781 ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac cccgtaattg
      841 attactatta ataactaatg catggcggta atacggttat ccacagaatc aggggataac
      901 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
      961 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     1021 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     1081 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     1141 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     1201 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     1261 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     1321 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     1381 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     1441 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1501 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     1561 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     1621 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     1681 tgaagtttta aatcaatcta aagtatatat gagtaacctg aggctatggc agggcctgcc
     1741 gccccgacgt tggctgcgag ccctgggcct tcacccgaac ttggggggtg gggtggggaa
     1801 aaggaagaaa cgcgggcgta ttggccccaa tggggtctcg gtggggtatc gacagagtgc
     1861 cagccctggg accgaacccc gcgtttatga acaaacgacc caacaccgtg cgttttattc
     1921 tgtcttttta ttgccgtcat agcgcgggtt ccttccggta ttgtctcctt ccgtgtttca
     1981 gttagcctcc ccctagggtg ggcgaagaac tccagcatga gatccccgcg ctggaggatc
     2041 atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa ggcggcggtg
     2101 gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc gaaccccaga
     2161 gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag
     2221 cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa
     2281 tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt
     2341 cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat
     2401 gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt gagcctggcg aacagttcgg
     2461 ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca
     2521 tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg
     2581 gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag
     2641 caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc
     2701 ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg
     2761 atagccgcgc tgcctcgtct tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa
     2821 aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg
     2881 tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt
     2941 gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga tcgatctttg
     3001 caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc agaggccgag
     3061 gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg agaatgggcg
     3121 gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac
     3181 taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac
     3241 ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg
     3301 gactttccac accctaactg acacacattc cacagctggt tctttccgcc tcaggactct
     3361 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     3421 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     3481 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3541 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3601 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3661 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     3721 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     3781 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     3841 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     3901 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttacgcctta agatacattg
     3961 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     4021 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     4081 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     4141 aaaacctcta caaatgtggt atggctgatt atgatctaga gtcgcggccg cgattacaat
     4201 ttggactttc cgcccttctt ggcctttatg aggatctctc tgatttttct tgcgtcgagt
     4261 tttccggtaa gacctttcgg tacttcgtcc acaaacacaa ctcctccgcg caactttttc
     4321 gcggttgtta cttgactggc gacgtaatcc acgatctctt tttccgtcat cgtctttccg
     4381 tgctccaaaa caacaacggc ggcgggaagt tcaccggcgt catcgtcggg aagacctgcc
     4441 acgcccgcgt cgaagatgtt ggggtgttgt aacaatatcg actccaattc agcgggggcc
     4501 acctggtatc ctttgtattt aattaaagac ttcaagcggt caactatgaa gaagtgttcg
     4561 tcttcgtccc agtaagctat gtctccagaa tgtagccatc catccttgtc aatcaaggcg
     4621 ttggtcgctt ccggattgtt tacataaccg gacataatca taggtcctct gacacataat
     4681 tcgcctctct gattaacgcc cagcgttttc ccggtatcca gatccacaac cttcgcttca
     4741 aaaaatggaa caactttacc gaccgcgccc ggtttatcat ccccctcggg tgtaatcaga
     4801 atagctgatg tagtctcagt gagcccatat ccttgtcgta tccctggaag atggaagcgt
     4861 tttgcaaccg cctccccgac ttctttcgaa agaggtgcgc ccccagaagc aatttcgtgt
     4921 aaattagata aatcgtattt gtcaatcaga gtgcttttgg cgaagaatga aaatagggtt
     4981 ggtactagca acgcactttg aattttgtaa tcctgaaggg atcgtaaaaa cagctcttct
     5041 tcaaatctat acattaagac gactcgaaat ccacatatca aatatccgag tgtagtaaac
     5101 attccaaaac cgtgatggaa tggaacaaca cttaaaatcg cagtatccgg aatgatttga
     5161 ttgccaaaaa taggatctct ggcatgcgag aatctgacgc aggcagttct atgcggaagg
     5221 gccacaccct taggtaaccc agtagatcca gaggagttca ttatcagtgc aattgttttg
     5281 tcacgatcaa aggactctgg tacaaaatcg tattcattaa aaccgggagg tagatgagat
     5341 gtgacgaacg tgtacatcga ctgaaatccc tggtaatccg ttttagaatc catgataata
     5401 attttctgga ttattggtaa ttttttttgc acgttcaaaa ttttttgcaa cccctttttg
     5461 gaaacaaaca ctacggtagg ctgcgaaatg ttcatactgt tgagcaattc acgttcatta
     5521 taaatgtcgt tcgcgggcgc aactgcaact ccgataaata acgcgcccaa caccggcata
     5581 aagaattgaa gagagttttc actgcatacg acgattctgt gatttgtatt cagcccatat
     5641 cgtttcatag cttctgccaa ccgaacggac atttcgaagt attccgcgta cgtgatgttc
     5701 acctcgatat gtgcatctgt aaaagcaatt gttccaggaa ccagggcgta tctcttcata
     5761 gccttatgca gttgctctcc agcggttcca tcctctagtg gatagaatgg cgccgggcct
     5821 ttctttatgt ttttggcgtc ttccataccg gaagggccgg gattctcctc cacgtcaccg
     5881 catgttagaa gacttcctct gccctcaccg gtcacattga tcctagcaga agcacaggct
     5941 gcagggtgac ggtccatccc gctctcctgg gcacaagaca tgggcagcgt gccatcatcc
     6001 tgcgccgcca ccgccggcgg gaagccatgg ctgatatctc gagatctggc gaaggcgatg
     6061 ggggtcttga aggcgtgctg gtactccacg atgcccagct cggtgttgct gtgcagctcc
     6121 tccacgcggc ggaaggcgaa catggggccc ccgttctgca ggatgctggg gtggatggcg
     6181 ctcttgaagt gcatgtggct gtccaccacg aagctgtagt agccgccgtc gcgcaggctg
     6241 aaggtgcggg cgaagctgcc caccagcacg ttatcgccca tggggtgcag gtgctccacg
     6301 gtggcgttgc tgcggatgat cttgtcggtg aagatcacgc tgtcctcggg gaagccggtg
     6361 cccaccacct tgaagtcgcc gatcacgcgg ccggcctcgt agcggtagct gaagctcacg
     6421 tgcagcacgc cgccgtcctc gtacttctcg atgcgggtgt tggtgtagcc gccgttgttg
     6481 atggcgtgca ggaaggggtt ctcgtagccg ctggggtagg tgccgaagtg gtagaagccg
     6541 tagcccatca cgtggctcag caggtagggg ctgaaggtca gggcgccttt ggtgctctt
//
