LOCUS       sequence-193122-        4432 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  H2B::linker::GFP (contain 3 introns)::3XFlag::TEV::Myc::tbb-2 3UTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4432)
  AUTHORS   Paix A, Folkmann A, Seydoux G
  TITLE     Precision genome editing using CRISPR-Cas9 and linear repair 
            templates in C. elegans.
  JOURNAL   Methods. 2017 May 15;121-122:86-93. doi: 
            10.1016/j.ymeth.2017.03.023. Epub 2017 Apr 7.
  PUBMED    28392263
REFERENCE   2  (bases 1 to 4432)
  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: "Methods."; 
            date: "2017-05-15"; volume: "121-122:86-93. doi"; pages: " 
            10.1016/j.ymeth.2017.03.023. Epub 2017 Apr 7"
COMMENT     Sequence Label: pAP608-1
FEATURES             Location/Qualifiers
     source          1..4432
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     347..366
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     600..617
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    734..755
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(770..775,776..793,794..800)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    808..824
                     /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)."
     primer_bind     813..835
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     832..848
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     832..848
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      complement(899..1222)
                     /gene="tbb-2"
                     /label=tbb-2 terminator
                     /note="transcription terminator for the C. elegans tubulin 
                     beta-2 gene"
     CDS             complement(1237..1266)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(1267..1287)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(1288..1353)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1394..1414)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     2162..2190
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(2664..2681)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2664..2680)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2673..2695)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2889..2908)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3008..3030
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3068..3086)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3154..3258
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3259..3327,3328..4119)
                     /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     complement(3477..3496)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4290..4432,1..446)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
       61 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
      121 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
      181 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
      241 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
      301 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
      361 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
      421 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
      481 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
      541 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
      601 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
      661 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
      721 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
      781 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
      841 gctatgacca tgattacgcc aagcttgcat gcctgcaggt cgactctaga ggatctgaga
      901 cttttttctt ggcggcacaa taaagtttcc tctttttgtt gggtcactca acgaacacaa
      961 tacaaatatt tgagcgctcg catttttctc aacctttcgg gtttaaagca cttcaaaatg
     1021 gaattgaaga gagattgtga aagtacatca gacagaatca cattttgata gcattcactt
     1081 cactcagatg caaacttttc agtgactata aaaagtcata aaataataac attaaaaaag
     1141 caaataaatt agcgagagaa ttttttgaca aaaagaaaga agagtgatag agaagaaggg
     1201 aatgcttgaa aggattttgc atttatcact agtttacaga tcctcttctg atatcagttt
     1261 ttgttctcct tggaagtaga ggttctcctt gtcatcgtca tccttgtaat cgatatcatg
     1321 atctttataa tcaccgtcat ggtctttgta gtctttgtat agttcgtcca tgccatgtgt
     1381 aatcccagca gctgttacaa actcaagaag gaccatgtgg tctctctttt cgttgggatc
     1441 tttcgaaagg gcagattgtg tggacaggta atggttgtct ggtaaaagga cagggccatc
     1501 gccaattgga gtattttgtt gataatggtc tgctagttga acgcttccat cttcaatgtt
     1561 gtgtctaatt ttgaagttct gaaaatttaa atcagattag ttagttagta aaatcatgtt
     1621 taaacttaca actttgattc cattcttttg tttgtctgcc atgatgtata cattgtgtga
     1681 gttatagttg tattccaatt tgtgtccaag aatgtttcca tcttctttaa aatcaatacc
     1741 ttttaactcg attctattaa caagggtatc accttcaaac ttgacttcag cacctgaaaa
     1801 tttaaatatg tatggttagt tagtaccgaa ctgtttaaac ttacgtgtct tgtagttccc
     1861 gtcatctttg aaaaatatag ttctttcctg tacataacct tcgggcatgg cactcttgaa
     1921 aaagtcatgc cgtttcatat gatctgggta tctcgagaag cattgaacac cataacagaa
     1981 agtagtgaca agtgttggct gaaaatttaa ataatcaggg ttagttagta tatatatgtt
     2041 taaacttacc catggaacag gtagttttcc agtagtgcaa ataaatttaa gggtaagttt
     2101 tccgtatgtt gcatcacctt caccctctcc actgacagaa aatttgtgcc cattaacatc
     2161 accatctaat tcaacaagaa ttgggacaac tccagtgaaa agttcttctc ctttactact
     2221 agttctagag cggccgccac cggtggagct ccaccgatcc cccgggcact tgctggaagt
     2281 gtacttggtg acggccttgg ttccctcaga cacggcgtgc ttggcaagtt ctcctgggag
     2341 aatcaaacgg acagcggttt gaatttcgcg ggatgagatc gttgagcgtt tgttgtaatg
     2401 agcaagacgg gaagcttccg aagcgatgcg ttcgaataca tcgttgacga aggagttcat
     2461 gatagacatg gccttggagg agactccggt gtctgggtga acttgcttga gaacacggta
     2521 gatgtagacg gagtacgatt ccttgcgggc atgacgtctc ttctttccgt cctttggctt
     2581 ggcaacgacg gtcttggcgg ccttcttggc tcccttggca gatggctttg gtggcatgat
     2641 ccccgggtac cgagctcgaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     2701 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     2761 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     2821 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc
     2881 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     2941 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3001 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     3061 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     3121 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3181 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3241 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     3301 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     3361 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     3421 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     3481 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     3541 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     3601 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     3661 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     3721 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     3781 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     3841 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     3901 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     3961 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     4021 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     4081 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     4141 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     4201 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4261 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4321 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4381 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa ta
//
