LOCUS       sequence-249895-        4947 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 4947)
  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 4947)
  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_17
FEATURES             Location/Qualifiers
     source          1..4947
                     /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"
     primer_bind     337..359
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     351..368
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     352..368
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     378..397
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        378..396
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(444..464)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(444..462)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(483..499)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(483..499)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(496..518)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    507..523
                     /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(531..537,538..555,556..561))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    576..597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        765..910
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          988..1120
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1103..1122
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1174..1183
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1180..1182,1183..1185,1186..1896)
                     /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(1225..1246)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1486..1505)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1833..1854
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2071..2192
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2082..2101)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2136..2155
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2440..2457)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2611..3199)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2691..2710)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3370..4161,4162..4230))
                     /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     3993..4012
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4231..4335)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4362..4817
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4449..4468)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4659..4680
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4895..4947,1..487))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
ORIGIN
        1 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta
       61 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc
      121 cccagtgcaa gtgcaggtgc cagaacattt ctctatcgat aggtaccgag ctcgagttgc
      181 cagggagacg ggcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
      241 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
      301 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
      361 cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacc gggccccccc
      421 tcgaggtcct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
      481 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
      541 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
      601 gcgttgcgct cactgcccgc tttccaccgg tcgtctcctg ttggctagcc cgggctcgag
      661 atctgcgatc taagtaagct tgctgcttca tccccgtggc ccgttgctcg cgtttgctgg
      721 cggtgtcccc ggaagaaata tatttgcatg tctttagttc tatgatgaca caaaccccgc
      781 ccagcgtctt gtcattggcg aattcgaaca cgcagatgca gtcggggcgg cgcggtccca
      841 ggtccacttc gcatattaag gtgacgcgtg tggcctcgaa caccgagcga ccctgcagcg
      901 acccgcttaa cagcgtcaac agcgtgccgc agatcccaag cttgcggccg cttaagctgc
      961 agaagttggt cgtgaggcac tgggcaggta agtatcaagg ttacaagaca ggtttaagga
     1021 gaccaataga aactgggctt gtcgagacag agaagactct tgcgtttctg ataggcacct
     1081 attggtctta ctgacatcca ctttgccttt ctctccacag gtgtccactc ccaggttcaa
     1141 tacagctctt aagcggccgc aagctagctt gccgccacca tggtgagcaa gggcgaggag
     1201 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1261 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1321 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac
     1381 ggcctgatgt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1441 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1501 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1561 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1621 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     1681 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1741 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccgcc
     1801 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1861 gccgggatca ctctcggcat ggacgagctg tacaagtgag cctctagact ccctcagcgg
     1921 cgcgccacca agtcagtgat tgcgcgagcg atgttggagt gctcctactg caggtacgaa
     1981 ggtcttatcg gatcgcccga acaccacaca gtgaccttga gatctcacgt gtctagagtc
     2041 ggggcggccg gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca
     2101 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     2161 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     2221 ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
     2281 gtaaaatcga taaggatccg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc
     2341 ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg
     2401 caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg
     2461 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     2521 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     2581 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2641 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2701 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2761 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag
     2821 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     2881 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2941 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3001 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     3061 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3121 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3181 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3241 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3301 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3361 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     3421 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     3481 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     3541 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3601 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3661 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3721 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3781 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3841 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     3901 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     3961 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4021 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     4081 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     4141 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     4201 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     4261 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     4321 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc
     4381 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4441 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4501 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4561 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4621 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4681 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     4741 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4801 attaacgctt acaatttgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     4861 ggtgcgggcc tcttcgctat tacgccagcc caagctacca tgataagtaa gtaatattaa
     4921 ggtacgggag gtacttggag cggccgc
//
