LOCUS       sequence-249891-        4942 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 4942)
  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 4942)
  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_4
FEATURES             Location/Qualifiers
     source          1..4942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1..49
                     /note="synthetic polyadenylation signal"
     misc_feature    63..154
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     103..122
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     337..359
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     351..368
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     352..368
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     378..397
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        378..396
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(444..464)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(444..462)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(483..499)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(483..499)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(496..518)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    507..523
                     /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(531..537,538..555,556..561))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    576..597
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        765..910
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          988..1120
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1103..1122
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1174..1183
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1180..1182,1183..1185,1186..1896)
                     /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(1225..1246)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1486..1505)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1833..1854
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2066..2187
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2077..2096)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2131..2150
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2435..2452)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2606..3194)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2686..2705)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3365..4156,4157..4225))
                     /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     3988..4007
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4226..4330)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4357..4812
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4444..4463)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4654..4675
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4890..4942,1..487))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
ORIGIN
        1 aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga atcgatagta
       61 ctaacatacg ctctccatca aaacaaaacg aaacaaaaca aactagcaaa ataggctgtc
      121 cccagtgcaa gtgcaggtgc cagaacattt ctctatcgat aggtaccgag ctcgagttgc
      181 cagggagacg ggcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
      241 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
      301 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
      361 cggccagtga gcgcgcgtaa tacgactcac tatagggcga attgggtacc gggccccccc
      421 tcgaggtcct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
      481 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
      541 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
      601 gcgttgcgct cactgcccgc tttccaccgg tcgtctcctg ttggctagcc cgggctcgag
      661 atctgcgatc taagtaagct tgctgcttca tccccgtggc ccgttgctcg cgtttgctgg
      721 cggtgtcccc ggaagaaata tatttgcatg tctttagttc tatgatgaca caaaccccgc
      781 ccagcgtctt gtcattggcg aattcgaaca cgcagatgca gtcggggcgg cgcggtccca
      841 ggtccacttc gcatattaag gtgacgcgtg tggcctcgaa caccgagcga ccctgcagcg
      901 acccgcttaa cagcgtcaac agcgtgccgc agatcccaag cttgcggccg cttaagctgc
      961 agaagttggt cgtgaggcac tgggcaggta agtatcaagg ttacaagaca ggtttaagga
     1021 gaccaataga aactgggctt gtcgagacag agaagactct tgcgtttctg ataggcacct
     1081 attggtctta ctgacatcca ctttgccttt ctctccacag gtgtccactc ccaggttcaa
     1141 tacagctctt aagcggccgc aagctagctt gccgccacca tggtgagcaa gggcgaggag
     1201 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1261 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1321 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac
     1381 ggcctgatgt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1441 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1501 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1561 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1621 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     1681 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1741 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccgcc
     1801 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1861 gccgggatca ctctcggcat ggacgagctg tacaagtgag cctctagact ccctcagcgg
     1921 cgcgcctgga tctgccgaca accaggttcg agcaaggacg caggttgtgt tgccggtaag
     1981 tcgacaatcg agcttgtgat cggtccttga agccagatct cacgtgtcta gagtcggggc
     2041 ggccggccgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact
     2101 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     2161 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     2221 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa
     2281 atcgataagg atccgtcgac cgatgccctt gagagccttc aacccagtca gctccttccg
     2341 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
     2401 cgtaggacag gtgccggcag cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2461 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2521 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2581 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2641 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2701 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2761 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     2821 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2881 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2941 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3001 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     3061 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3121 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3181 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3241 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3301 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3361 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3421 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3481 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3541 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3601 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3661 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3721 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3781 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3841 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3901 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3961 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4021 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4081 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4141 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4201 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4261 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4321 gggttccgcg cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa
     4381 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     4441 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4501 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4561 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4621 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4681 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     4741 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4801 cgcttacaat ttgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     4861 gggcctcttc gctattacgc cagcccaagc taccatgata agtaagtaat attaaggtac
     4921 gggaggtact tggagcggcc gc
//
