LOCUS       sequence-258394-        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_24
FEATURES             Location/Qualifiers
     source          1..4945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     55..77
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     69..86
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     70..86
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     96..115
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        96..114
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(162..182)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(162..180)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(201..217)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(201..217)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(214..236)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    225..241
                     /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(249..255,256..273,274..279))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    294..315
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        483..628
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          706..838
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     821..840
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      892..901
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(898..900,901..903,904..1614)
                     /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(943..964)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1204..1223)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1551..1572
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1787..1908
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1798..1817)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1852..1871
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2156..2173)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2327..2915)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2407..2426)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3086..3877,3878..3946))
                     /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     3709..3728
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3947..4051)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4078..4533
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4165..4184)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4375..4396
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4611..4945,1..205))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4664..4712
                     /note="synthetic polyadenylation signal"
     misc_feature    4726..4817
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4766..4785
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
       61 cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat
      121 tgggtaccgg gccccccctc gaggtcctcc agcttttgtt ccctttagtg agggttaatt
      181 gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
      241 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
      301 agctaactca cattaattgc gttgcgctca ctgcccgctt tccaccggtc gtctcctgtt
      361 ggctagcccg ggctcgagat ctgcgatcta agtaagcttg ctgcttcatc cccgtggccc
      421 gttgctcgcg tttgctggcg gtgtccccgg aagaaatata tttgcatgtc tttagttcta
      481 tgatgacaca aaccccgccc agcgtcttgt cattggcgaa ttcgaacacg cagatgcagt
      541 cggggcggcg cggtcccagg tccacttcgc atattaaggt gacgcgtgtg gcctcgaaca
      601 ccgagcgacc ctgcagcgac ccgcttaaca gcgtcaacag cgtgccgcag atcccaagct
      661 tgcggccgct taagctgcag aagttggtcg tgaggcactg ggcaggtaag tatcaaggtt
      721 acaagacagg tttaaggaga ccaatagaaa ctgggcttgt cgagacagag aagactcttg
      781 cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct ctccacaggt
      841 gtccactccc aggttcaata cagctcttaa gcggccgcaa gctagcttgc cgccaccatg
      901 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
      961 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1021 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1081 gtgaccacct tcggctacgg cctgatgtgc ttcgcccgct accccgacca catgaagcag
     1141 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1201 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     1261 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     1321 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     1381 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     1441 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     1501 ctgagctacc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     1561 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtgagcc
     1621 tctagactcc ctcagcggcg cgcctgtcga agctaaaagt gctcgagccc aataccattt
     1681 cggaatctcg cgtggcgtta gtatgccact cgatggtagg ggaacgcaga atgccagatc
     1741 tcacgtgtct agagtcgggg cggccggccg cttcgagcag acatgataag atacattgat
     1801 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     1861 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     1921 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa
     1981 aacctctaca aatgtggtaa aatcgataag gatccgtcga ccgatgccct tgagagcctt
     2041 caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac
     2101 tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc
     2161 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2221 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2281 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     2341 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2401 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2461 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     2521 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     2581 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     2641 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     2701 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     2761 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     2821 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     2881 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     2941 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3001 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3061 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3121 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3181 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3241 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3301 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3361 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3421 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3481 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     3541 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     3601 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     3661 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     3721 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     3781 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     3841 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     3901 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     3961 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4021 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     4081 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     4141 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     4201 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     4261 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     4321 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     4381 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     4441 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     4501 cgaattttaa caaaatatta acgcttacaa tttgccattc gccattcagg ctgcgcaact
     4561 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagcccaag ctaccatgat
     4621 aagtaagtaa tattaaggta cgggaggtac ttggagcggc cgcaataaaa tatctttatt
     4681 ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gtactaacat acgctctcca
     4741 tcaaaacaaa acgaaacaaa acaaactagc aaaataggct gtccccagtg caagtgcagg
     4801 tgccagaaca tttctctatc gataggtacc gagctcgagt tgccagggag acgggcgccg
     4861 ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     4921 cgggcctctt cgctattacg ccagc
//
