LOCUS       sequence-258995-        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_Cerulean_Nanotag_80
FEATURES             Location/Qualifiers
     source          1..4947
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    15..36
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        204..349
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          427..559
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     542..561
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      613..622
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(619..621,622..624,625..1338)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(664..685)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(925..944)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1272..1293
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1510..1631
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1521..1540)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1575..1594
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1879..1896)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2050..2638)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2130..2149)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2809..3600,3601..3669))
                     /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     3432..3451
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3670..3774)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3801..4256
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3888..3907)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4098..4119
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4334..4873)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPWN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4387..4435
                     /note="synthetic polyadenylation signal"
     misc_feature    4449..4540
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4489..4508
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4723..4745
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4737..4754
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4738..4754
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4764..4783
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4764..4782
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4830..4850)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4830..4848)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4869..4885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4869..4885)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4882..4904)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4893..4909
                     /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(4917..4923,4924..4941,4942..4947))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
       61 ttccaccggt cgtctcagga ccgctagccc gggctcgaga tctgcgatct aagtaagctt
      121 gctgcttcat ccccgtggcc cgttgctcgc gtttgctggc ggtgtccccg gaagaaatat
      181 atttgcatgt ctttagttct atgatgacac aaaccccgcc cagcgtcttg tcattggcga
      241 attcgaacac gcagatgcag tcggggcggc gcggtcccag gtccacttcg catattaagg
      301 tgacgcgtgt ggcctcgaac accgagcgac cctgcagcga cccgcttaac agcgtcaaca
      361 gcgtgccgca gatcccaagc ttgcggccgc ttaagctgca gaagttggtc gtgaggcact
      421 gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg
      481 tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac tgacatccac
      541 tttgcctttc tctccacagg tgtccactcc caggttcaat acagctctta agcggccgca
      601 agctagcttg ccgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      661 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      721 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      781 cccgtgccct ggcccaccct cgtgaccacc ctgacctggg gcgtgcagtg cttcgcccgc
      841 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      901 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
      961 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1021 ggcaacatcc tggggcacaa gctggagtac aacgccatca gcgacaacgt ctatatcacc
     1081 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac
     1141 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1201 ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag
     1261 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1321 gacgagctgt acaagtgagc ctctagactc cctcagcggc gcgcccgtga gcattacgga
     1381 gccgactaca ctcaggcagg agccgcttaa ggagctgctc ctctaacgaa cggcaagatt
     1441 ggcagcgtag atgcgcctag atctcacgtg tctagagtcg gggcggccgg ccgcttcgag
     1501 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     1561 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     1621 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
     1681 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatccgt
     1741 cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg
     1801 actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg
     1861 gcagcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     1921 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     1981 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2041 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2101 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2161 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2221 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2281 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2341 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2401 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     2461 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     2521 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2581 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2641 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     2701 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     2761 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     2821 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     2881 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     2941 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     3001 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     3061 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     3121 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     3181 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     3241 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     3301 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     3361 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     3421 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     3481 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     3541 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     3601 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     3661 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     3721 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     3781 tccccgaaaa gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3841 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3901 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3961 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     4021 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4081 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     4141 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     4201 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttgcca
     4261 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     4321 acgccagccc aagctaccat gataagtaag taatattaag gtacgggagg tacttggagc
     4381 ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaatcg
     4441 atagtactaa catacgctct ccatcaaaac aaaacgaaac aaaacaaact agcaaaatag
     4501 gctgtcccca gtgcaagtgc aggtgccaga acatttctct atcgataggt accgagctcg
     4561 agttccatgg gagacgggcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
     4621 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     4681 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     4741 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtaccgggc
     4801 cccccctcga ggtcctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     4861 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4921 atacgagccg gaagcataaa gtgtaaa
//
