LOCUS       sequence-258396-        4949 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 4949)
  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 4949)
  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_32
FEATURES             Location/Qualifiers
     source          1..4949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        154..299
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          377..509
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     492..511
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      563..572
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(569..571,572..574,575..1285)
                     /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(614..635)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(875..894)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1222..1243
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1462..1583
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1473..1492)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1527..1546
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1831..1848)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2002..2590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2082..2101)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2761..3552,3553..3621))
                     /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     3384..3403
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3622..3726)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3753..4208
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3840..3859)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4050..4071
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4286..4825)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4339..4387
                     /note="synthetic polyadenylation signal"
     misc_feature    4401..4492
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4441..4460
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4675..4697
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4689..4706
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4690..4706
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4716..4735
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4716..4734
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4782..4802)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4782..4800)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4821..4837)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4821..4837)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4834..4856)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4845..4861
                     /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(4869..4875,4876..4893,4894..4899))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4914..4935
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 actgcccgct ttccaccggt cgtctcctgt tggctagccc gggctcgaga tctgcgatct
       61 aagtaagctt gctgcttcat ccccgtggcc cgttgctcgc gtttgctggc ggtgtccccg
      121 gaagaaatat atttgcatgt ctttagttct atgatgacac aaaccccgcc cagcgtcttg
      181 tcattggcga attcgaacac gcagatgcag tcggggcggc gcggtcccag gtccacttcg
      241 catattaagg tgacgcgtgt ggcctcgaac accgagcgac cctgcagcga cccgcttaac
      301 agcgtcaaca gcgtgccgca gatcccaagc ttgcggccgc ttaagctgca gaagttggtc
      361 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa
      421 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac
      481 tgacatccac tttgcctttc tctccacagg tgtccactcc caggttcaat acagctctta
      541 agcggccgca agctagcttg ccgccaccat ggtgagcaag ggcgaggagc tgttcaccgg
      601 ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc
      661 cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac
      721 cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgatgtg
      781 cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga
      841 aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc
      901 cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt
      961 caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt
     1021 ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa
     1081 catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga
     1141 cggccccgtg ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga
     1201 ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac
     1261 tctcggcatg gacgagctgt acaagtgagc ctctagactc cctcagcggc gcgccgctct
     1321 ccagaaactt cgcaaacacc tatgcctaac gccagccgaa tcctgcagtc cgttacctag
     1381 tcgaaaggca ttctgggagc atcgcgtcac agatctcacg tgtctagagt cggggcggcc
     1441 ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
     1501 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     1561 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     1621 agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg
     1681 ataaggatcc gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg
     1741 gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta
     1801 ggacaggtgc cggcagcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     1861 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     1921 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     1981 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     2041 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2101 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2161 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     2221 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     2281 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2341 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2401 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     2461 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     2521 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     2581 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     2641 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     2701 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     2761 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     2821 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     2881 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     2941 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     3001 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     3061 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     3121 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     3181 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     3241 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     3301 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     3361 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     3421 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     3481 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     3541 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     3601 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     3661 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     3721 tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc
     3781 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3841 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3901 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3961 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4021 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4081 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4141 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     4201 tacaatttgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     4261 ctcttcgcta ttacgccagc ccaagctacc atgataagta agtaatatta aggtacggga
     4321 ggtacttgga gcggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt
     4381 ttgtgtgaat cgatagtact aacatacgct ctccatcaaa acaaaacgaa acaaaacaaa
     4441 ctagcaaaat aggctgtccc cagtgcaagt gcaggtgcca gaacatttct ctatcgatag
     4501 gtaccgagct cgagttgcca gggagacggg cgccgctaca gggcgcgtcc cattcgccat
     4561 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     4621 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     4681 cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat
     4741 tgggtaccgg gccccccctc gaggtcctcc agcttttgtt ccctttagtg agggttaatt
     4801 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4861 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4921 agctaactca cattaattgc gttgcgctc
//
