LOCUS       sequence-153088-        4069 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 4069)
  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 4069)
  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-2A-TurboGFP-PEST-PuroR
FEATURES             Location/Qualifiers
     source          1..4069
                     /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             200..253
                     /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             260..955
                     /codon_start=1
                     /product="improved green fluorescent protein from 
                     Pontellina plumata (Evdokimov et al., 2006)"
                     /label=TurboGFP
                     /note="mammalian codon-optimized"
                     /translation="MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTPEQGRMTNKM
                     KSTKGALTFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVL
                     HVSFSYRYEAGRVIGDFKVMGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNDLDGSFTR
                     TFSLRDGGYYSSVVDSHMHFKSAIHPSILQNGGPMFAFRRVEEDHSNTELGIVEYQHAF
                     KTPDADAGEE"
     CDS             1100..1153
                     /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"
     polyA_signal    1777..1898
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1788..1807)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1842..1861
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2146..2163)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2317..2905)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2397..2416)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3105..3896,3897..3965))
                     /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 tagctagcgg gccagtaccc
      181 aaaaagcggg gggagcggcg agggcagagg aagtctgcta acatgcggtg acgtcgagga
      241 gaatcctggc ccaggatcca tggagagcga cgagagcggc ctgcccgcca tggagatcga
      301 gtgccgcatc accggcaccc tgaacggcgt ggagttcgag ctggtgggcg gcggagaggg
      361 cacccccgag cagggccgca tgaccaacaa gatgaagagc accaaaggcg ccctgacctt
      421 cagcccctac ctgctgagcc acgtgatggg ctacggcttc taccacttcg gcacctaccc
      481 cagcggctac gagaacccct tcctgcacgc catcaacaac ggcggctaca ccaacacccg
      541 catcgagaag tacgaggacg gcggcgtgct gcacgtgagc ttcagctacc gctacgaggc
      601 cggccgcgtg atcggcgact tcaaggtgat gggcaccggc ttccccgagg acagcgtgat
      661 cttcaccgac aagatcatcc gcagcaacgc caccgtggag cacctgcacc ccatgggcga
      721 taacgatctg gatggcagct tcacccgcac cttcagcctg cgcgacggcg gctactacag
      781 ctccgtggtg gacagccaca tgcacttcaa gagcgccatc caccccagca tcctgcagaa
      841 cggcggcccc atgttcgcct tccgccgcgt ggaggaggat cacagcaaca ccgagctggg
      901 catcgtggag taccagcacg ccttcaagac cccggatgca gatgccggtg aagaaagatc
      961 tcgagatatc agccatggct tcccgccggc ggtggcggcg caggatgatg gcacgctgcc
     1021 catgtcttgt gcccaggaga gcgggatgga ccgtcaccct gcagcctgtg cttctgctag
     1081 gatcaatgtg tccggaggag agggcagagg aagtctgcta acatgcggtg acgtcgagga
     1141 gaatcctggc ccaatgactg aatacaagcc tactgtcagg ttggctacaa gagacgacgt
     1201 tcctagagcc gtgagaactc tggctgcagc cttcgccgac taccccgcca cgagacacac
     1261 cgttgaccca gatcggcata ttgagagagt gactgaactg caggagctgt ttcttacaag
     1321 agttggcctc gacataggca aggtgtgggt ggcggacgac ggcgccgccg tggccgtctg
     1381 gaccactccc gaatcagttg aggctggcgc cgtattcgct gagatcggcc cgagaatggc
     1441 tgagctcagc gggagtaggc tcgcggcaca gcagcaaatg gagggactgc tggcaccaca
     1501 caggcccaaa gaacccgcct ggttcctggc aaccgtcggt gtatctcccg atcatcaggg
     1561 gaaaggtctg ggctctgccg tagtgctccc tggcgtggag gcagctgaga gagcaggagt
     1621 acctgccttc ttggagacct ccgctccaag gaatcttccc ttctatgaac ggttgggctt
     1681 caccgtgaca gccgacgtgg aagtccccga aggcccccgc acttggtgca tgacgaggaa
     1741 gcctggagcg tgagggcccg cttcgagcag acatgataag atacattgat gagtttggac
     1801 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     1861 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     1921 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
     1981 aatgtggtaa aatcgataaa gatccgtcga ccgatgccct tgagagcctt caacccagtc
     2041 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     2101 atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc tcactgactc
     2161 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2221 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2281 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2341 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2401 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2461 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2521 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2581 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2641 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2701 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     2761 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2821 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2881 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2941 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3001 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3061 aacttggtct gacagcggcc gcaaatgcta aaccactgca gtggttacca gtgcttgatc
     3121 agtgaggcac cgatctcagc gatctgccta tttcgttcgt ccatagtggc ctgactcccc
     3181 gtcgtgtaga tcactacgat tcgtgagggc ttaccatcag gccccagcgc agcaatgatg
     3241 ccgcgagagc cgcgttcacc ggcccccgat ttgtcagcaa tgaaccagcc agcagggagg
     3301 gccgagcgaa gaagtggtcc tgctactttg tccgcctcca tccagtctat gagctgctgt
     3361 cgtgatgcta gagtaagaag ttcgccagtg agtagtttcc gaagagttgt ggccattgct
     3421 actggcatcg tggtatcacg ctcgtcgttc ggtatggctt cgttcaactc tggttcccag
     3481 cggtcaagcc gggtcacatg atcacccata ttatgaagaa atgcagtcag ctccttaggg
     3541 cctccgatcg ttgtcagaag taagttggcc gcggtgttgt cgctcatggt aatggcagca
     3601 ctacacaatt ctcttaccgt catgccatcc gtaagatgct tttccgtgac cggcgagtac
     3661 tcaaccaagt cgttttgtga gtagtgtata cggcgaccaa gctgctcttg cccggcgtct
     3721 atacgggaca acaccgcgcc acatagcagt actttgaaag tgctcatcat cgggaatcgt
     3781 tcttcggggc ggaaagactc aaggatcttg ccgctattga gatccagttc gatatagccc
     3841 actcttgcac ccagttgatc ttcagcatct tttactttca ccagcgtttc ggggtgtgca
     3901 aaaacaggca agcaaaatgc cgcaaagaag ggaatgagtg cgacacgaaa atgttggatg
     3961 ctcatactcg tcctttttca atattattga agcatttatc agggttacta gtacgtctct
     4021 caaggataag taagtaatat taaggtacgg gaggtattgg acaggccgc
//
