LOCUS       sequence-249899-        4945 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 4945)
  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 4945)
  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_24
FEATURES             Location/Qualifiers
     source          1..4945
                     /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    2069..2190
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2080..2099)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2134..2153
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2438..2455)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2609..3197)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2689..2708)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3368..4159,4160..4228))
                     /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     3991..4010
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4229..4333)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4360..4815
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4447..4466)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4657..4678
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4893..4945,1..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 cgcgcctgtc gaagctaaaa gtgctcgagc ccaataccat ttcggaatct cgcgtggcgt
     1981 tagtatgcca ctcgatggta ggggaacgca gaatgccaga tctcacgtgt ctagagtcgg
     2041 ggcggccggc cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     2101 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     2161 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     2221 caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     2281 aaaatcgata aggatccgtc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
     2341 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
     2401 actcgtagga caggtgccgg cagcgctctt ccgcttcctc gctcactgac tcgctgcgct
     2461 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2521 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2581 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2641 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2701 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2761 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
     2821 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2881 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2941 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3001 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     3061 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3121 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3181 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3241 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3301 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3361 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     3421 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     3481 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3541 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3601 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3661 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3721 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3781 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3841 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3901 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3961 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4021 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4081 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4141 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     4201 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     4261 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     4321 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt agcggcgcat
     4381 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     4441 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4501 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4561 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4621 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4681 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     4741 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     4801 taacgcttac aatttgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     4861 tgcgggcctc ttcgctatta cgccagccca agctaccatg ataagtaagt aatattaagg
     4921 tacgggaggt acttggagcg gccgc
//
