LOCUS       sequence-261244-        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_Citrine_Nanotag_19
FEATURES             Location/Qualifiers
     source          1..4945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..96
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     88..105
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     89..105
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     115..134
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        115..133
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(181..201)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(181..199)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(220..236)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(220..236)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(233..255)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    244..260
                     /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(268..274,275..292,293..298))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    313..334
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        502..647
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          725..857
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     840..859
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      911..920
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(917..919,920..922,923..1633)
                     /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(962..983)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1223..1242)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1570..1591
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1806..1927
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1817..1836)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1871..1890
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2175..2192)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2346..2934)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2426..2445)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3105..3896,3897..3965))
                     /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     3728..3747
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3966..4070)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4097..4552
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4184..4203)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4394..4415
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4630..4945,1..224))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4683..4731
                     /note="synthetic polyadenylation signal"
     misc_feature    4745..4836
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4785..4804
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
       61 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac
      121 gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcctcca gcttttgttc
      181 cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg
      241 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
      301 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
      361 ccaccggtcg tctcctgttg gctagcccgg gctcgagatc tgcgatctaa gtaagcttgc
      421 tgcttcatcc ccgtggcccg ttgctcgcgt ttgctggcgg tgtccccgga agaaatatat
      481 ttgcatgtct ttagttctat gatgacacaa accccgccca gcgtcttgtc attggcgaat
      541 tcgaacacgc agatgcagtc ggggcggcgc ggtcccaggt ccacttcgca tattaaggtg
      601 acgcgtgtgg cctcgaacac cgagcgaccc tgcagcgacc cgcttaacag cgtcaacagc
      661 gtgccgcaga tcccaagctt gcggccgctt aagctgcaga agttggtcgt gaggcactgg
      721 gcaggtaagt atcaaggtta caagacaggt ttaaggagac caatagaaac tgggcttgtc
      781 gagacagaga agactcttgc gtttctgata ggcacctatt ggtcttactg acatccactt
      841 tgcctttctc tccacaggtg tccactccca ggttcaatac agctcttaag cggccgcaag
      901 ctagcttgcc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      961 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     1021 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1081 cgtgccctgg cccaccctcg tgaccacctt cggctacggc ctgatgtgct tcgcccgcta
     1141 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1201 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1261 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1321 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1381 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1441 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1501 gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa
     1561 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1621 cgagctgtac aagtgagcct ctagactccc tcagcggcgc gcccaatggg tgtattgtgg
     1681 gttgccgatt tacggcgagt cttccgacgg atatggacat tgccggaaca gcctgtactg
     1741 gagcgacctt acggagatct cacgtgtcta gagtcggggc ggccggccgc ttcgagcaga
     1801 catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg
     1861 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     1921 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga
     1981 ggttttttaa agcaagtaaa acctctacaa atgtggtaaa atcgataagg atccgtcgac
     2041 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
     2101 tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag
     2161 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2221 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2281 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2341 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2401 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     2461 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     2521 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2581 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     2641 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2701 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2761 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2821 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2881 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2941 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3001 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3061 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3121 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3181 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3241 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3301 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3361 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3421 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3481 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3541 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3601 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3661 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3721 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3781 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3841 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3901 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3961 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4021 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4081 cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4141 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4201 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4261 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     4321 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4381 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     4441 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     4501 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttgccattcg
     4561 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     4621 cagcccaagc taccatgata agtaagtaat attaaggtac gggaggtact tggagcggcc
     4681 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag
     4741 tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca aaataggctg
     4801 tccccagtgc aagtgcaggt gccagaacat ttctctatcg ataggtaccg agctcgagtt
     4861 gccagggaga cgggcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg
     4921 ttgggaaggg cgatcggtgc gggcc
//
