LOCUS       sequence-257644-        4937 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 4937)
  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 4937)
  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_117
FEATURES             Location/Qualifiers
     source          1..4937
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..51
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     43..60
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     44..60
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     70..89
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        70..88
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(136..156)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(136..154)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(175..191)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(175..191)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(188..210)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    199..215
                     /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(223..229,230..247,248..253))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    268..289
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        457..602
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          680..812
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     795..814
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      866..875
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             872..1582
                     /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(1017..1035)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1285..1304
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1753..1874
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1764..1783)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1818..1837
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2122..2139)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2293..2881)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2373..2392)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3052..3843,3844..3912))
                     /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     3675..3694
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3913..4017)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4044..4499
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4131..4150)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4341..4362
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4577..4937,1..179))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLLHN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4630..4678
                     /note="synthetic polyadenylation signal"
     misc_feature    4692..4783
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4732..4751
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
       61 gagcgcgcgt aatacgactc actatagggc gaattgggta ccgggccccc cctcgaggtc
      121 ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata
      181 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
      241 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
      301 ctcactgccc gctttccacc ggtcgtctca cggtggctag cccgggctcg agatctgcga
      361 tctaagtaag cttgctgctt catccccgtg gcccgttgct cgcgtttgct ggcggtgtcc
      421 ccggaagaaa tatatttgca tgtctttagt tctatgatga cacaaacccc gcccagcgtc
      481 ttgtcattgg cgaattcgaa cacgcagatg cagtcggggc ggcgcggtcc caggtccact
      541 tcgcatatta aggtgacgcg tgtggcctcg aacaccgagc gaccctgcag cgacccgctt
      601 aacagcgtca acagcgtgcc gcagatccca agcttgcggc cgcttaagct gcagaagttg
      661 gtcgtgaggc actgggcagg taagtatcaa ggttacaaga caggtttaag gagaccaata
      721 gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac ctattggtct
      781 tactgacatc cactttgcct ttctctccac aggtgtccac tcccaggttc aatacagctc
      841 ttaagcggcc gcaagctagc ttgccgccac catggtgagc aagggcgagg aggataacat
      901 ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
      961 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     1021 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     1081 catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     1141 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     1201 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     1261 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     1321 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
     1381 gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
     1441 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa
     1501 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     1561 catggacgag ctgtacaagt gagcctctag actccctcag cggcgcgcca cggacggatg
     1621 tgcggtagaa tcatgcttga cgagtgagga tggaccgacc caaacgacag agggttagca
     1681 ccgctacctc ctaagcgctt tagatctcac gtgtctagag tcggggcggc cggccgcttc
     1741 gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     1801 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     1861 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     1921 tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc
     1981 cgtcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc
     2041 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
     2101 ccggcagcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     2161 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     2221 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     2281 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     2341 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     2401 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     2461 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     2521 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     2581 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     2641 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     2701 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     2761 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     2821 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     2881 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     2941 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3001 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     3061 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3121 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3181 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     3241 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     3301 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     3361 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     3421 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     3481 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     3541 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     3601 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     3661 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     3721 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     3781 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     3841 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     3901 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     3961 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     4021 atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     4081 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4141 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4201 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4261 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4321 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4381 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4441 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttg
     4501 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     4561 attacgccag cccaagctac catgataagt aagtaatatt aaggtacggg aggtacttgg
     4621 agcggccgca ataaaatatc tttattttca ttacatctgt gtgttggttt tttgtgtgaa
     4681 tcgatagtac taacatacgc tctccatcaa aacaaaacga aacaaaacaa actagcaaaa
     4741 taggctgtcc ccagtgcaag tgcaggtgcc agaacatttc tctatcgata ggtaccgagc
     4801 tcgagttgtg caggagacgg gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc
     4861 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     4921 ggggatgtgc tgcaagg
//
