LOCUS       sequence-258398-        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_51
FEATURES             Location/Qualifiers
     source          1..4946
                     /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..1299)
                     /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(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    1473..1594
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1484..1503)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1538..1557
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1842..1859)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2013..2601)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2093..2112)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2772..3563,3564..3632))
                     /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     3395..3414
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3633..3737)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3764..4219
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3851..3870)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4061..4082
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4297..4836)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4350..4398
                     /note="synthetic polyadenylation signal"
     misc_feature    4412..4503
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4452..4471
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4686..4708
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4700..4717
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4701..4717
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4727..4746
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4727..4745
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4793..4813)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4793..4811)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4832..4848)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4832..4848)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4845..4867)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4856..4872
                     /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(4880..4886,4887..4904,4905..4910))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4925..4946
                     /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 mygktggcta 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 caccttcggc
      781 tacggcctga tgtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
      841 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
      901 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
      961 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     1021 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     1081 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1141 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc
     1201 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1261 gccgccggga tcactctcgg catggacgag ctgtacaagt gagcctctag actccctcag
     1321 cggcgcgccg atctgcggtt gccgaggtat cttccgggta ttatgcgcca ttgcgccacg
     1381 gactagcatc ctgctagatt cgcgtcttga tacgggccta aagatctcac gtgtctagag
     1441 tcggggcggc cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac
     1501 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     1561 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     1621 gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     1681 tggtaaaatc gataaggatc cgtcgaccga tgcccttgag agccttcaac ccagtcagct
     1741 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca
     1801 tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt cctcgctcac tgactcgctg
     1861 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     1921 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     1981 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2041 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2101 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2161 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2221 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2281 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     2341 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     2401 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     2461 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     2521 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     2581 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     2641 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     2701 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     2761 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     2821 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     2881 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     2941 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3001 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3061 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3121 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3181 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3241 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     3301 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     3361 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     3421 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     3481 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     3541 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     3601 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     3661 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     3721 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgcgcc ctgtagcggc
     3781 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3841 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3901 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3961 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4021 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4081 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     4141 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4201 atattaacgc ttacaatttg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     4261 tcggtgcggg cctcttcgct attacgccag cccaagctac catgataagt aagtaatatt
     4321 aaggtacggg aggtacttgg agcggccgca ataaaatatc tttattttca ttacatctgt
     4381 gtgttggttt tttgtgtgaa tcgatagtac taacatacgc tctccatcaa aacaaaacga
     4441 aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag tgcaggtgcc agaacatttc
     4501 tctatcgata ggtaccgagc tcgagttgcc agggagacgg gcgccgctac agggcgcgtc
     4561 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     4621 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     4681 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
     4741 atagggcgaa ttgggtaccg ggccccccct cgaggtcctc cagcttttgt tccctttagt
     4801 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     4861 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     4921 cctaatgagt gagctaactc acatta
//
