LOCUS       sequence-257582-        4950 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 4950)
  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 4950)
  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_92
FEATURES             Location/Qualifiers
     source          1..4950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        168..313
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          391..523
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     506..525
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      577..586
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(583..585,586..588,589..1302)
                     /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(628..649)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(889..908)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1236..1257
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1477..1598
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1488..1507)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1542..1561
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1846..1863)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2017..2605)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2097..2116)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2776..3567,3568..3636))
                     /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     3399..3418
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3637..3741)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3768..4223
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3855..3874)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4065..4086
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4301..4840)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPWN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4354..4402
                     /note="synthetic polyadenylation signal"
     misc_feature    4416..4507
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4456..4475
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4690..4712
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4704..4721
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4705..4721
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4731..4750
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4731..4749
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4797..4817)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4797..4815)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4836..4852)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4836..4852)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4849..4871)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4860..4876
                     /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(4884..4890,4891..4908,4909..4914))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4929..4950
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 attgcgttgc gctcactgcc cgctttccac cggtcgtctc aggaccgcta gcccgggctc
       61 gagatctgcg atctaagtaa gcttgctgct tcatccccgt ggcccgttgc tcgcgtttgc
      121 tggcggtgtc cccggaagaa atatatttgc atgtctttag ttctatgatg acacaaaccc
      181 cgcccagcgt cttgtcattg gcgaattcga acacgcagat gcagtcgggg cggcgcggtc
      241 ccaggtccac ttcgcatatt aaggtgacgc gtgtggcctc gaacaccgag cgaccctgca
      301 gcgacccgct taacagcgtc aacagcgtgc cgcagatccc aagcttgcgg ccgcttaagc
      361 tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa
      421 ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca
      481 cctattggtc ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccaggtt
      541 caatacagct cttaagcggc cgcaagctag cttgccgcca ccatggtgag caagggcgag
      601 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      661 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
      721 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
      781 tggggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
      841 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
      901 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
      961 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacgcc
     1021 atcagcgaca acgtctatat caccgccgac aagcagaaga acggcatcaa ggccaacttc
     1081 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1141 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     1201 aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1261 gccgccggga tcactctcgg catggacgag ctgtacaagt gagcctctag actccctcag
     1321 cggcgcgcct tgctgtctgt accgggtggt accacggtca ttatcggcag ctgtttactt
     1381 cacgcaaagt acagtccgct ttccggtaca agccagtcta atggtagatc tcacgtgtct
     1441 agagtcgggg cggccggccg cttcgagcag acatgataag atacattgat gagtttggac
     1501 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     1561 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     1621 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
     1681 aatgtggtaa aatcgataag gatccgtcga ccgatgccct tgagagcctt caacccagtc
     1741 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     1801 atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc tcactgactc
     1861 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     1921 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     1981 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2041 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2101 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2161 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2221 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2281 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2341 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2401 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     2461 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2521 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2581 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2641 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2701 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2761 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2821 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     2881 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     2941 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3001 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3061 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3121 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3181 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3241 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3301 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3361 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3421 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3481 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3541 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3601 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     3661 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3721 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag
     3781 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3841 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3901 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3961 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     4021 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4081 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     4141 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     4201 caaaatatta acgcttacaa tttgccattc gccattcagg ctgcgcaact gttgggaagg
     4261 gcgatcggtg cgggcctctt cgctattacg ccagcccaag ctaccatgat aagtaagtaa
     4321 tattaaggta cgggaggtac ttggagcggc cgcaataaaa tatctttatt ttcattacat
     4381 ctgtgtgttg gttttttgtg tgaatcgata gtactaacat acgctctcca tcaaaacaaa
     4441 acgaaacaaa acaaactagc aaaataggct gtccccagtg caagtgcagg tgccagaaca
     4501 tttctctatc gataggtacc gagctcgagt tccatgggag acgggcgccg ctacagggcg
     4561 cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     4621 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     4681 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact
     4741 cactataggg cgaattgggt accgggcccc ccctcgaggt cctccagctt ttgttccctt
     4801 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4861 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4921 ggtgcctaat gagtgagcta actcacatta
//
