LOCUS       sequence-258393-        4949 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 4949)
  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 4949)
  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_70
FEATURES             Location/Qualifiers
     source          1..4949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        126..271
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          349..481
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     464..483
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      535..544
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(541..543,544..546,547..1260)
                     /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(586..607)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(847..866)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1194..1215
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1434..1555
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1445..1464)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1499..1518
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1803..1820)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1974..2562)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2054..2073)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2733..3524,3525..3593))
                     /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     3356..3375
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3594..3698)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3725..4180
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3812..3831)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4022..4043
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4258..4797)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPWN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4311..4359
                     /note="synthetic polyadenylation signal"
     misc_feature    4373..4464
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4413..4432
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4647..4669
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4661..4678
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4662..4678
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4688..4707
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4688..4706
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4754..4774)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4754..4772)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4793..4809)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4793..4809)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4806..4828)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4817..4833
                     /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(4841..4847,4848..4865,4866..4871))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4886..4907
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gaccgctagc ccgggctcga gatctgcgat ctaagtaagc ttgctgcttc atccccgtgg
       61 cccgttgctc gcgtttgctg gcggtgtccc cggaagaaat atatttgcat gtctttagtt
      121 ctatgatgac acaaaccccg cccagcgtct tgtcattggc gaattcgaac acgcagatgc
      181 agtcggggcg gcgcggtccc aggtccactt cgcatattaa ggtgacgcgt gtggcctcga
      241 acaccgagcg accctgcagc gacccgctta acagcgtcaa cagcgtgccg cagatcccaa
      301 gcttgcggcc gcttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag
      361 gttacaagac aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc
      421 ttgcgtttct gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca
      481 ggtgtccact cccaggttca atacagctct taagcggccg caagctagct tgccgccacc
      541 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      601 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
      661 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      721 ctcgtgacca ccctgacctg gggcgtgcag tgcttcgccc gctaccccga ccacatgaag
      781 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
      841 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
      901 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
      961 aagctggagt acaacgccat cagcgacaac gtctatatca ccgccgacaa gcagaagaac
     1021 ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1081 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1141 tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1201 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga
     1261 gcctctagac tccctcagcg gcgcgccaac acactggacg cagcctccgc agtgtggtag
     1321 acgccgtgga tatgtcttcg caaatacgga gccagatcgt gagcagtaag cttgtctatg
     1381 gtagatctca cgtgtctaga gtcggggcgg ccggccgctt cgagcagaca tgataagata
     1441 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     1501 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     1561 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag
     1621 caagtaaaac ctctacaaat gtggtaaaat cgataaggat ccgtcgaccg atgcccttga
     1681 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     1741 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctcttccgct
     1801 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1861 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1921 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1981 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2041 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2101 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2161 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2221 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2281 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2341 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2401 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2461 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2521 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2581 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2641 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2701 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2761 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2821 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     2881 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     2941 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3001 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3061 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3121 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3181 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3241 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3301 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3361 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3421 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3481 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     3541 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     3601 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3661 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3721 cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     3781 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     3841 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     3901 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     3961 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     4021 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     4081 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     4141 tttaacgcga attttaacaa aatattaacg cttacaattt gccattcgcc attcaggctg
     4201 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gcccaagcta
     4261 ccatgataag taagtaatat taaggtacgg gaggtacttg gagcggccgc aataaaatat
     4321 ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta ctaacatacg
     4381 ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc cccagtgcaa
     4441 gtgcaggtgc cagaacattt ctctatcgat aggtaccgag ctcgagttcc atgggagacg
     4501 ggcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg
     4561 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
     4621 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
     4681 gcgcgcgtaa tacgactcac tatagggcga attgggtacc gggccccccc tcgaggtcct
     4741 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
     4801 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4861 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4921 cactgcccgc tttccaccgg tcgtctcag
//
