LOCUS       sequence-257572-        4948 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 4948)
  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 4948)
  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_58
FEATURES             Location/Qualifiers
     source          1..4948
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        124..269
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          347..479
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     462..481
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      533..542
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(539..541,542..544,545..1255)
                     /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(584..605)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(845..864)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1192..1213
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1431..1552
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1442..1461)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1496..1515
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1800..1817)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1971..2559)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2051..2070)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2730..3521,3522..3590))
                     /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     3353..3372
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3591..3695)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3722..4177
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3809..3828)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4019..4040
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4255..4794)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4308..4356
                     /note="synthetic polyadenylation signal"
     misc_feature    4370..4461
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4410..4429
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4644..4666
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4658..4675
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4659..4675
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4685..4704
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4685..4703
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4751..4771)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4751..4769)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4790..4806)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4790..4806)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4803..4825)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4814..4830
                     /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(4838..4844,4845..4862,4863..4868))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4883..4904
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tggctagccc gggctcgaga tctgcgatct aagtaagctt gctgcttcat ccccgtggcc
       61 cgttgctcgc gtttgctggc ggtgtccccg gaagaaatat atttgcatgt ctttagttct
      121 atgatgacac aaaccccgcc cagcgtcttg tcattggcga attcgaacac gcagatgcag
      181 tcggggcggc gcggtcccag gtccacttcg catattaagg tgacgcgtgt ggcctcgaac
      241 accgagcgac cctgcagcga cccgcttaac agcgtcaaca gcgtgccgca gatcccaagc
      301 ttgcggccgc ttaagctgca gaagttggtc gtgaggcact gggcaggtaa gtatcaaggt
      361 tacaagacag gtttaaggag accaatagaa actgggcttg tcgagacaga gaagactctt
      421 gcgtttctga taggcaccta ttggtcttac tgacatccac tttgcctttc tctccacagg
      481 tgtccactcc caggttcaat acagctctta agcggccgca agctagcttg ccgccaccat
      541 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
      601 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
      661 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
      721 cgtgaccacc ttcggctacg gcctgatgtg cttcgcccgc taccccgacc acatgaagca
      781 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
      841 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
      901 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
      961 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     1021 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     1081 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1141 cctgagctac cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1201 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtgagc
     1261 ctctagactc cctcagcggc gcgcctgatc agggcatgct atgcggagtg tgccgtagaa
     1321 cgagaccctt gcgggtttag ccgacacacc tcccgaatcc cagcgtaaac gttacggaaa
     1381 gatctcacgt gtctagagtc ggggcggccg gccgcttcga gcagacatga taagatacat
     1441 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     1501 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     1561 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa
     1621 gtaaaacctc tacaaatgtg gtaaaatcga taaggatccg tcgaccgatg cccttgagag
     1681 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     1741 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc ttccgcttcc
     1801 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     1861 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     1921 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     1981 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2041 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2101 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2161 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2221 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2281 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2341 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2401 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2461 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2521 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2581 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2641 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2701 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     2761 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     2821 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     2881 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     2941 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3001 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3061 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3121 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3181 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3241 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3301 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3361 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3421 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3481 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3541 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3601 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     3661 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     3721 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3781 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3841 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3901 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3961 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4021 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4081 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4141 aacgcgaatt ttaacaaaat attaacgctt acaatttgcc attcgccatt caggctgcgc
     4201 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagcc caagctacca
     4261 tgataagtaa gtaatattaa ggtacgggag gtacttggag cggccgcaat aaaatatctt
     4321 tattttcatt acatctgtgt gttggttttt tgtgtgaatc gatagtacta acatacgctc
     4381 tccatcaaaa caaaacgaaa caaaacaaac tagcaaaata ggctgtcccc agtgcaagtg
     4441 caggtgccag aacatttctc tatcgatagg taccgagctc gagttgccag ggagacgggc
     4501 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
     4561 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     4621 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg
     4681 cgcgtaatac gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcctcca
     4741 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     4801 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4861 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4921 tgcccgcttt ccaccggtcg tctcctgt
//
