LOCUS       sequence-257735-        4941 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 4941)
  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 4941)
  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_Cherry_Nanotag_109
FEATURES             Location/Qualifiers
     source          1..4941
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        104..249
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          327..459
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     442..461
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      513..522
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             519..1229
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(664..682)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     932..951
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1404..1525
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1415..1434)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1469..1488
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1773..1790)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1944..2532)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2024..2043)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2703..3494,3495..3563))
                     /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     3326..3345
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3564..3668)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3695..4150
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3782..3801)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3992..4013
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4228..4767)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLLHN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4281..4329
                     /note="synthetic polyadenylation signal"
     misc_feature    4343..4434
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4383..4402
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4617..4639
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4631..4648
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4632..4648
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4658..4677
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4658..4676
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4724..4744)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4724..4742)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4763..4779)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4763..4779)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4776..4798)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4787..4803
                     /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(4811..4817,4818..4835,4836..4841))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4856..4877
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tctgcgatct aagtaagctt gctgcttcat ccccgtggcc cgttgctcgc gtttgctggc
       61 ggtgtccccg gaagaaatat atttgcatgt ctttagttct atgatgacac aaaccccgcc
      121 cagcgtcttg tcattggcga attcgaacac gcagatgcag tcggggcggc gcggtcccag
      181 gtccacttcg catattaagg tgacgcgtgt ggcctcgaac accgagcgac cctgcagcga
      241 cccgcttaac agcgtcaaca gcgtgccgca gatcccaagc ttgcggccgc ttaagctgca
      301 gaagttggtc gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag
      361 accaatagaa actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta
      421 ttggtcttac tgacatccac tttgcctttc tctccacagg tgtccactcc caggttcaat
      481 acagctctta agcggccgca agctagcttg ccgccaccat ggtgagcaag ggcgaggagg
      541 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
      601 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
      661 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
      721 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
      781 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
      841 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
      901 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct
      961 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     1021 agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg
     1081 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct
     1141 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     1201 ccggcggcat ggacgagctg tacaagtgag cctctagact ccctcagcgg cgcgccatcg
     1261 gagcatatcg tgcttgtcga ttaggagcct gtctgcttga accgtttggg tgggcgatac
     1321 ttcagtcgca tgccgtttga caagagacac ttagatctca cgtgtctaga gtcggggcgg
     1381 ccggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     1441 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     1501 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     1561 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat
     1621 cgataaggat ccgtcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     1681 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     1741 taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     1801 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     1861 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1921 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1981 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     2041 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     2101 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     2161 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     2221 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     2281 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     2341 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     2401 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2461 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2521 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2581 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2641 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2701 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2761 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     2821 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     2881 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     2941 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     3001 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     3061 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     3121 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     3181 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     3241 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     3301 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     3361 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3421 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     3481 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3541 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     3601 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     3661 gttccgcgca catttccccg aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc
     3721 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3781 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     3841 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     3901 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     3961 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4021 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4081 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     4141 cttacaattt gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
     4201 gcctcttcgc tattacgcca gcccaagcta ccatgataag taagtaatat taaggtacgg
     4261 gaggtacttg gagcggccgc aataaaatat ctttattttc attacatctg tgtgttggtt
     4321 ttttgtgtga atcgatagta ctaacatacg ctctccatca aaacaaaacg aaacaaaaca
     4381 aactagcaaa ataggctgtc cccagtgcaa gtgcaggtgc cagaacattt ctctatcgat
     4441 aggtaccgag ctcgagttgt gcaggagacg ggcgccgcta cagggcgcgt cccattcgcc
     4501 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     4561 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     4621 gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga
     4681 attgggtacc gggccccccc tcgaggtcct ccagcttttg ttccctttag tgagggttaa
     4741 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4801 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4861 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccaccgg tcgtctcacg
     4921 gtggctagcc cgggctcgag a
//
