LOCUS       sequence-343260-        3908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPaint gene tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3908)
  AUTHORS   Schmid-Burgk JL, Honing K, Ebert TS, Hornung V
  TITLE     CRISPaint allows modular base-specific gene tagging using a 
            ligase-4-dependent mechanism.
  JOURNAL   Nat Commun. 2016 Jul 28;7:12338. doi: 10.1038/ncomms12338.
  PUBMED    27465542
REFERENCE   2  (bases 1 to 3908)
  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: "Nat 
            Commun."; date: "2016-07-28"; volume: "7:12338. doi"; pages: " 
            10.1038/ncomms12338"
COMMENT     Sequence Label: pCRISPaint-mNeon-PuroR
FEATURES             Location/Qualifiers
     source          1..3908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1..49
                     /note="synthetic polyadenylation signal"
     misc_feature    63..154
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     103..122
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     CDS             227..928
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEEL
                     NLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
                     SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTF
                     KWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKA
                     FTDVMGMDELYK"
     regulatory      476..485
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             938..991
                     /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"
     CDS             992..1591
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(992..1011)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1488..1508
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1616..1737
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1627..1646)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1681..1700
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1985..2002)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2156..2744)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2236..2255)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2944..3735,3736..3804))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta
       61 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc
      121 cccagtgcaa gtgcaggtgc cagaacattt ctcttaatga tagctaggat cgctagcggg
      181 ccagtaccca aaaagcgggg ggtctggtgg cagtggaggg ggatccgtga gcaagggcga
      241 ggaggataac gcctctctcc cagcgacaca tgagttacac atctttggct ccatcaacgg
      301 tgtggacttt gacatggtgg gtcagggcac cggcaatcca aatgatggtt atgaggagtt
      361 aaacctgaag tccaccaagg gtgacctcca gttctccccc tggattctgg tccctcatat
      421 cgggtatggc ttccatcagt acctgcccta ccctgacggg atgtcgcctt tccaggccgc
      481 catggtagat ggctccggat accaagtcca tcgcacaatg cagtttgaag atggtgcctc
      541 ccttactgtt aactaccgct acacctacga gggaagccac atcaaaggag aggcccaggt
      601 gaaggggact ggtttccctg ctgacggtcc tgtgatgacc aactcgctga ccgctgcgga
      661 ctggtgcagg tcgaagaaga cttaccccaa cgacaaaacc atcatcagta cctttaagtg
      721 gagttacacc actggaaatg gcaagcgcta ccggagcact gcgcggacca cctacacctt
      781 tgccaagcca atggcggcta actatctgaa gaaccagccg atgtacgtgt tccgtaagac
      841 ggagctcaag cactccaaga ccgagctcaa cttcaaggag tggcaaaagg cctttaccga
      901 tgtgatgggc atggacgagc tgtacaaggg atccggagag ggcagaggaa gtctgctaac
      961 atgcggtgac gtcgaggaga atcctggccc aatgaccgag tacaagccca cggtgcgcct
     1021 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta
     1081 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca
     1141 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg
     1201 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga
     1261 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga
     1321 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt
     1381 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc
     1441 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt
     1501 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac
     1561 ctggtgcatg acccgcaagc ccggtgcctg agcggcccgc ttcgagcaga catgataaga
     1621 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     1681 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     1741 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     1801 agcaagtaaa acctctacaa atgtggtaaa atcgataagg atccgtcgac cgatgccctt
     1861 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     1921 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctcttccg
     1981 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     2041 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     2101 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2161 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2221 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2281 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2341 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2401 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     2461 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     2521 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     2581 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     2641 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     2701 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     2761 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     2821 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     2881 tctaaagtat atatgagtaa acttggtctg acagcggccg caaatgctaa accactgcag
     2941 tggttaccag tgcttgatca gtgaggcacc gatctcagcg atctgcctat ttcgttcgtc
     3001 catagtggcc tgactccccg tcgtgtagat cactacgatt cgtgagggct taccatcagg
     3061 ccccagcgca gcaatgatgc cgcgagagcc gcgttcaccg gcccccgatt tgtcagcaat
     3121 gaaccagcca gcagggaggg ccgagcgaag aagtggtcct gctactttgt ccgcctccat
     3181 ccagtctatg agctgctgtc gtgatgctag agtaagaagt tcgccagtga gtagtttccg
     3241 aagagttgtg gccattgcta ctggcatcgt ggtatcacgc tcgtcgttcg gtatggcttc
     3301 gttcaactct ggttcccagc ggtcaagccg ggtcacatga tcacccatat tatgaagaaa
     3361 tgcagtcagc tccttagggc ctccgatcgt tgtcagaagt aagttggccg cggtgttgtc
     3421 gctcatggta atggcagcac tacacaattc tcttaccgtc atgccatccg taagatgctt
     3481 ttccgtgacc ggcgagtact caaccaagtc gttttgtgag tagtgtatac ggcgaccaag
     3541 ctgctcttgc ccggcgtcta tacgggacaa caccgcgcca catagcagta ctttgaaagt
     3601 gctcatcatc gggaatcgtt cttcggggcg gaaagactca aggatcttgc cgctattgag
     3661 atccagttcg atatagccca ctcttgcacc cagttgatct tcagcatctt ttactttcac
     3721 cagcgtttcg gggtgtgcaa aaacaggcaa gcaaaatgcc gcaaagaagg gaatgagtgc
     3781 gacacgaaaa tgttggatgc tcatactcgt cctttttcaa tattattgaa gcatttatca
     3841 gagttactag tacgtctctc aaggataagt aagtaatatt aaggtacggg aggtattgga
     3901 caggccgc
//
