LOCUS       sequence-250021-        6429 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Modified pTK plasmid containing chicken EdnrB E2 enhancer driving 
            expression of full length Chicken Tfap2B in frame with an Avi tag at
            the N terminus and FLAG-tag and Citrine at the C-terminus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6429)
  AUTHORS   Ling ITC, Sauka-Spengler T
  TITLE     Early chromatin shaping predetermines multipotent vagal neural crest
            into neural, neuronal and mesenchymal lineages.
  JOURNAL   Nat Cell Biol. 2019 Dec;21(12):1504-1517. doi: 
            10.1038/s41556-019-0428-9. Epub 2019 Dec 2.
  PUBMED    31792380
REFERENCE   2  (bases 1 to 6429)
  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"; doi: 
            "10.1038/s41556-019-0428-9"; journalName: "Nat Cell Biol"; date: 
            "2019-12"; volume: "21"; issue: "12"; pages: "1504-1517"
COMMENT     Sequence Label: pTK-EdnrbE2-AVI-Tfap2B-tev-FLAG-2A-Citrine
FEATURES             Location/Qualifiers
     source          1..6429
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    101..149
                     /note="synthetic polyadenylation signal"
     misc_feature    163..254
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     promoter        928..1073
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          1151..1283
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1266..1285
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1337..1346
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1352..1396
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             1403..1423
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1427..1450
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2813..2866
                     /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             join(2882..2884,2885..2887,2888..3598)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2927..2948)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3188..3207)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3535..3556
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3653..3774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3664..3683)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3718..3737
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4022..4039)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4193..4781)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4273..4292)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4952..5743,5744..5812))
                     /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     5575..5594
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5813..5917)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5944..6399
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6031..6050)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6241..6262
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gcccaagcta ccatgataag
       61 taagtaatat taaggtacgg gaggtacttg gagcggccgc aataaaatat ctttattttc
      121 attacatctg tgtgttggtt ttttgtgtga atcgatagta ctaacatacg ctctccatca
      181 aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc cccagtgcaa gtgcaggtgc
      241 cagaacattt ctctatcgat aggtaccgag ctcgagttgc cagtgcttgt aggaccgctc
      301 aagtgcttgc agctcacaat taccctgctg gcagttgtaa ccgccgcact aggactaagt
      361 cgcttgtgtc ttgtcattac aatgcttcct tttgtccgat ggctgcaaat gcttgacagt
      421 cattagaaag tcatccaagg aaatacttcc tttccatgta caagatgctt gtagtggatt
      481 tttaacactg agctttctca gtaactcaga tagcattagc aaaagaatag tttcttacag
      541 cctgcctctg cccttaaggt tcttttataa agagaacaga agctctttgt tgttactcct
      601 tcacacgtta ttaacagagg tttaacagaa agaagagggg cttgttatag tccgctgcaa
      661 tttgtctttt tcagttcatt cggatctcca tcatgtccaa ccagaagacg gattaaccca
      721 gatccagggt ggctgcttcc atactaaaca gaaaatgaat tttaaaaagt cagacagtgt
      781 aatctctctc tgccaagaac ctgttggcta gcccgggctc gagatctgcg atctaagtaa
      841 gcttgctgct tcatccccgt ggcccgttgc tcgcgtttgc tggcggtgtc cccggaagaa
      901 atatatttgc atgtctttag ttctatgatg acacaaaccc cgcccagcgt cttgtcattg
      961 gcgaattcga acacgcagat gcagtcgggg cggcgcggtc ccaggtccac ttcgcatatt
     1021 aaggtgacgc gtgtggcctc gaacaccgag cgaccctgca gcgacccgct taacagcgtc
     1081 aacagcgtgc cgcagatccc aagcttgcgg ccgcttaagc tgcagaagtt ggtcgtgagg
     1141 cactgggcag gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg
     1201 cttgtcgaga cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat
     1261 ccactttgcc tttctctcca caggtgtcca ctcccaggtt caatacagct cttaagcggc
     1321 cgcaagctag cttgccgcca ccatggctgg tggcctgaat gacatctttg aggcccagaa
     1381 gatcgagtgg catgagggag gagagaacct gtacttccag ggaggagact acaaggacga
     1441 cgacgacaag gatatgctct ggaaactggt tgaaaatgtc aagtacgaag atatctatga
     1501 ggaccggcat gatggagtgc ccagccacag ttccaggctg tcccagctgg gatccgtctc
     1561 ccagggaccc tactccagcg ctcctccgct ctctcacacc ccgtcctcgg atttccagcc
     1621 gccttatttc ccacccccct accagcctct tccttaccac caaagccagg acccttactc
     1681 ccatgtcaat gacccctact ccctaaaccc cttgcatcaa ccccagcagc acccctgggg
     1741 acagaggcag aggcaagagg tgggatcaga caccgggtca ctgctgccac agcctcgggc
     1801 cgccctgccc cagctctcgg gactggaccc caggcgggac taccactcgg tacggcggcc
     1861 agatgtcctg ctgcactcag ctcaccatgg ccttgactcc ggcatggggg acagcctttc
     1921 cctgcacggc atcgggcacc cggggatgga ggaggtccag tcagttgaag atgccaataa
     1981 cagcggtatg aacttattgg accagtctgt cattaaaaaa gttcctgtcc ctccaaaatc
     2041 cgttacttct ttgatgatga ataaagatgg cttcctgggt ggaatttccg tcaacaccgg
     2101 agaggtgttc tgctctgtcc ccggccgttt gtctttgctt agttcaactt ccaagtacaa
     2161 agtaacagtg ggagaagttc aaaggcgcct ttctcccccc gagtgtctga acgcatccct
     2221 gctaggagga gtgctgagaa gagccaaatc gaaaaacggg gggagatctt tgcgagaaag
     2281 gctagaaaaa atcggtttga atttaccagc cggcaggcgt aaggctgcaa atgtcacttt
     2341 actcacctcc ctggtggaag gtgaggccgt tcatttagcc cgggattttg ggtacatctg
     2401 cgaaacggag ttcccagcca aggctgtttc tgaatacctg aacagacagc acacagaccc
     2461 cagcgacctc cactccagga aaaacatgct gcttgctaca aagcaacttt gtaaagaatt
     2521 tacagatctc ttggcccagg acaggacacc cattggaaac agccggccca cccctatttt
     2581 ggaaccaggc attcagagct gcttgactca cttcagcctc atcactcacg gctttggggc
     2641 tcccgctatc tgcgccgccc tcacggccct ccaaaactac ctgactgaag ctctcaaagg
     2701 catggacaag atgttcttga acaacaacac tgctaacagg cacacatctg gcgaaggacc
     2761 aggtagtaaa actggagaca aggaagagaa acacagaaaa atcggatcag gagagggcag
     2821 gggaagtctt ctaacatgcg gggacgtgga ggaaaatccc gggcccggcg gatccggagg
     2881 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     2941 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     3001 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     3061 cctcgtgacc accttcggct acggcctgat gtgcttcgcc cgctaccccg accacatgaa
     3121 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     3181 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     3241 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     3301 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     3361 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     3421 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     3481 ctacctgagc taccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     3541 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     3601 aagcggccgc gactctagag tcggggcggc cggccgcttc gagcagacat gataagatac
     3661 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     3721 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     3781 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     3841 aagtaaaacc tctacaaatg tggtaaaatc gataaggatc cgtcgaccga tgcccttgag
     3901 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     3961 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt
     4021 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4081 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4141 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4201 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4261 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4321 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4381 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4441 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4501 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4561 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4621 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4681 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4741 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     4801 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4861 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4921 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4981 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5041 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5101 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5161 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5221 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5281 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5341 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5401 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5461 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5521 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     5581 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     5641 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     5701 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     5761 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     5821 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5881 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5941 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     6001 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     6061 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     6121 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     6181 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     6241 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     6301 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     6361 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttg ccattcgcca ttcaggctgc
     6421 gcaactgtt
//
