LOCUS       sequence-258389-        4946 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 4946)
  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 4946)
  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_Citrine_Nanotag_5
FEATURES             Location/Qualifiers
     source          1..4946
                     /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..1335)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     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    1509..1630
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1520..1539)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1574..1593
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1878..1895)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2049..2637)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2129..2148)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2808..3599,3600..3668))
                     /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     3431..3450
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3669..3773)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3800..4255
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3887..3906)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4097..4118
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4333..4872)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4386..4434
                     /note="synthetic polyadenylation signal"
     misc_feature    4448..4539
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4488..4507
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4722..4744
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4736..4753
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4737..4753
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4763..4782
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4763..4781
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4829..4849)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4829..4847)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4868..4884)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4868..4884)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4881..4903)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4892..4908
                     /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(4916..4922,4923..4940,4941..4946))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
       61 ttccaccggt cgtctcctgt tggctagccc 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 ttcggctacg gcctgatgtg 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 aactacaaca gccacaacgt ctatatcatg
     1081 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1141 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1201 ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga ccccaacgag
     1261 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1321 gacgagctgt acaagtgagc ctctagactc cctcagcggc gcgccgttgc gttccaaacg
     1381 tcgtggagct ccctgtcaag acacgaaccg ctaacgccac cttagtccgt gaagcatgat
     1441 acgcgacata ccccaggaga tctcacgtgt ctagagtcgg ggcggccggc cgcttcgagc
     1501 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     1561 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     1621 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     1681 ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatcgata aggatccgtc
     1741 gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga
     1801 ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg
     1861 cagcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     1921 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     1981 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2041 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2101 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2161 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2221 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     2281 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2341 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2401 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2461 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     2521 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     2581 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     2641 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     2701 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     2761 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     2821 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     2881 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     2941 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3001 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3061 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3121 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     3181 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3241 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3301 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3361 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3421 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3481 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     3541 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3601 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     3661 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     3721 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     3781 ccccgaaaag tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     3841 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     3901 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     3961 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     4021 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4081 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     4141 gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     4201 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttgccat
     4261 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     4321 cgccagccca agctaccatg ataagtaagt aatattaagg tacgggaggt acttggagcg
     4381 gccgcaataa aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga
     4441 tagtactaac atacgctctc catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg
     4501 ctgtccccag tgcaagtgca ggtgccagaa catttctcta tcgataggta ccgagctcga
     4561 gttgccaggg agacgggcgc cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa
     4621 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     4681 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     4741 aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg gtaccgggcc
     4801 ccccctcgag gtcctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     4861 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4921 tacgagccgg aagcataaag tgtaaa
//
