LOCUS       sequence-257576-        4945 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 4945)
  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 4945)
  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_61
FEATURES             Location/Qualifiers
     source          1..4945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          78..210
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     193..212
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      264..273
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(270..272,273..275,276..989)
                     /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(315..336)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(576..595)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     923..944
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1159..1280
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1170..1189)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1224..1243
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1528..1545)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1699..2287)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1779..1798)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2458..3249,3250..3318))
                     /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     3081..3100
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3319..3423)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3450..3905
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3537..3556)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3747..3768
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3983..4522)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPWN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4036..4084
                     /note="synthetic polyadenylation signal"
     misc_feature    4098..4189
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4138..4157
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4372..4394
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4386..4403
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4387..4403
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4413..4432
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4413..4431
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4479..4499)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4479..4497)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4518..4534)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4518..4534)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4531..4553)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4542..4558
                     /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(4566..4572,4573..4590,4591..4596))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4611..4632
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4800..4945
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
ORIGIN
        1 cagcgtcaac agcgtgccgc agatcccaag cttgcggccg cttaagctgc agaagttggt
       61 cgtgaggcac tgggcaggta agtatcaagg ttacaagaca ggtttaagga gaccaataga
      121 aactgggctt gtcgagacag agaagactct tgcgtttctg ataggcacct attggtctta
      181 ctgacatcca ctttgccttt ctctccacag gtgtccactc ccaggttcaa tacagctctt
      241 aagcggccgc aagctagctt gccgccacca tggtgagcaa gggcgaggag ctgttcaccg
      301 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
      361 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
      421 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctgg ggcgtgcagt
      481 gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
      541 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
      601 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
      661 tcaaggagga cggcaacatc ctggggcaca agctggagta caacgccatc agcgacaacg
      721 tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag atccgccaca
      781 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
      841 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccaag ctgagcaaag
      901 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
      961 ctctcggcat ggacgagctg tacaagtgag cctctagact ccctcagcgg cgcgccagga
     1021 catgagtact tcccgagaga gcgatgtcaa ccgaatcctg acgggacttc gtaattcgga
     1081 ccaggtgttc ggagcttgtg aggagataga tctcacgtgt ctagagtcgg ggcggccggc
     1141 cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     1201 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     1261 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     1321 gggaggtgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatcgata
     1381 aggatccgtc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
     1441 cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga
     1501 caggtgccgg cagcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     1561 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     1621 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     1681 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     1741 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     1801 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     1861 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     1921 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     1981 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2041 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2101 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     2161 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     2221 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     2281 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     2341 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     2401 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     2461 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     2521 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     2581 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     2641 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     2701 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     2761 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     2821 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     2881 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     2941 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     3001 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     3061 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     3121 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     3181 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     3241 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     3301 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     3361 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     3421 gcgcacattt ccccgaaaag tgccacctga cgcgccctgt agcggcgcat taagcgcggc
     3481 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3541 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3601 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3661 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3721 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3781 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3841 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     3901 aatttgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     3961 ttcgctatta cgccagccca agctaccatg ataagtaagt aatattaagg tacgggaggt
     4021 acttggagcg gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg
     4081 tgtgaatcga tagtactaac atacgctctc catcaaaaca aaacgaaaca aaacaaacta
     4141 gcaaaatagg ctgtccccag tgcaagtgca ggtgccagaa catttctcta tcgataggta
     4201 ccgagctcga gttccatggg agacgggcgc cgctacaggg cgcgtcccat tcgccattca
     4261 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     4321 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     4381 gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg
     4441 gtaccgggcc ccccctcgag gtcctccagc ttttgttccc tttagtgagg gttaattgcg
     4501 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4561 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4621 taactcacat taattgcgtt gcgctcactg cccgctttcc accggtcgtc tcaggaccgc
     4681 tagcccgggc tcgagatctg cgatctaagt aagcttgctg cttcatcccc gtggcccgtt
     4741 gctcgcgttt gctggcggtg tccccggaag aaatatattt gcatgtcttt agttctatga
     4801 tgacacaaac cccgcccagc gtcttgtcat tggcgaattc gaacacgcag atgcagtcgg
     4861 ggcggcgcgg tcccaggtcc acttcgcata ttaaggtgac gcgtgtggcc tcgaacaccg
     4921 agcgaccctg cagcgacccg cttaa
//
