LOCUS       sequence-257555-        4942 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Enhancer cloning nanotag reporter vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4942)
  AUTHORS   Williams RM, Candido-Ferreira I, Repapi E, Gavriouchkina D, 
            Senanayake U, Ling ITC, Telenius J, Taylor S, Hughes J, 
            Sauka-Spengler T
  TITLE     Reconstruction of the Global Neural Crest Gene Regulatory Network In
            Vivo.
  JOURNAL   Dev Cell. 2019 Oct 21;51(2):255-276.e7. doi: 
            10.1016/j.devcel.2019.10.003.
  PUBMED    31639368
REFERENCE   2  (bases 1 to 4942)
  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: 
            "10.1016/j.devcel.2019.10"; journalName: "Dev Cell"; date: 
            "2019-10-21- 21"; volume: "51"; issue: "2"; pages: "255-276.e7"
COMMENT     Sequence Label: pTK_BsmBI_LacZ_Citrine_Nanotag_4
FEATURES             Location/Qualifiers
     source          1..4942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     4..26
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     18..35
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     19..35
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     45..64
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        45..63
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(111..131)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(111..129)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(150..166)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(150..166)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(163..185)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    174..190
                     /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)."
     promoter        complement(join(198..204,205..222,223..228))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    243..264
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        432..577
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          655..787
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     770..789
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      841..850
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(847..849,850..852,853..1563)
                     /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(892..913)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1153..1172)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1500..1521
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1733..1854
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1744..1763)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1798..1817
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2102..2119)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2273..2861)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2353..2372)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3032..3823,3824..3892))
                     /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     3655..3674
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3893..3997)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4024..4479
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4111..4130)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4321..4342
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4557..4942,1..154))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4610..4658
                     /note="synthetic polyadenylation signal"
     misc_feature    4672..4763
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4712..4731
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat
       61 agggcgaatt gggtaccggg ccccccctcg aggtcctcca gcttttgttc cctttagtga
      121 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
      181 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
      241 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccaccggtcg
      301 tctcctgttg gctagcccgg gctcgagatc tgcgatctaa gtaagcttgc tgcttcatcc
      361 ccgtggcccg ttgctcgcgt ttgctggcgg tgtccccgga agaaatatat ttgcatgtct
      421 ttagttctat gatgacacaa accccgccca gcgtcttgtc attggcgaat tcgaacacgc
      481 agatgcagtc ggggcggcgc ggtcccaggt ccacttcgca tattaaggtg acgcgtgtgg
      541 cctcgaacac cgagcgaccc tgcagcgacc cgcttaacag cgtcaacagc gtgccgcaga
      601 tcccaagctt gcggccgctt aagctgcaga agttggtcgt gaggcactgg gcaggtaagt
      661 atcaaggtta caagacaggt ttaaggagac caatagaaac tgggcttgtc gagacagaga
      721 agactcttgc gtttctgata ggcacctatt ggtcttactg acatccactt tgcctttctc
      781 tccacaggtg tccactccca ggttcaatac agctcttaag cggccgcaag ctagcttgcc
      841 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      901 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      961 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1021 cccaccctcg tgaccacctt cggctacggc ctgatgtgct tcgcccgcta ccccgaccac
     1081 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1141 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1201 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1261 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1321 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1381 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1441 aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1501 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1561 aagtgagcct ctagactccc tcagcggcgc gcctggatct gccgacaacc aggttcgagc
     1621 aaggacgcag gttgtgttgc cggtaagtcg acaatcgagc ttgtgatcgg tccttgaagc
     1681 cagatctcac gtgtctagag tcggggcggc cggccgcttc gagcagacat gataagatac
     1741 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     1801 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     1861 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     1921 aagtaaaacc tctacaaatg tggtaaaatc gataaggatc cgtcgaccga tgcccttgag
     1981 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     2041 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt
     2101 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     2161 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2221 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2281 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2341 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2401 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2461 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2521 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2581 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2641 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2701 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2761 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2821 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2881 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2941 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3001 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3061 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3121 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3181 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3241 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3301 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3361 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3421 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3481 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3541 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3601 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3661 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3721 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3781 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3841 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3901 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     3961 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4021 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     4081 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     4141 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     4201 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     4261 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     4321 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     4381 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     4441 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttg ccattcgcca ttcaggctgc
     4501 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag cccaagctac
     4561 catgataagt aagtaatatt aaggtacggg aggtacttgg agcggccgca ataaaatatc
     4621 tttattttca ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac taacatacgc
     4681 tctccatcaa aacaaaacga aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag
     4741 tgcaggtgcc agaacatttc tctatcgata ggtaccgagc tcgagttgcc agggagacgg
     4801 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     4861 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     4921 ttaagttggg taacgccagg gt
//
