LOCUS       sequence-257574-        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_Cerulean_Nanotag_60
FEATURES             Location/Qualifiers
     source          1..4945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        185..330
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          408..540
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     523..542
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      594..603
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(600..602,603..605,606..1319)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(645..666)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(906..925)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1253..1274
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1489..1610
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1500..1519)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1554..1573
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1858..1875)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2029..2617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2109..2128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2788..3579,3580..3648))
                     /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     3411..3430
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3649..3753)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3780..4235
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3867..3886)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4077..4098
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4313..4852)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPWN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4366..4414
                     /note="synthetic polyadenylation signal"
     misc_feature    4428..4519
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4468..4487
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4702..4724
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4716..4733
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4717..4733
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4743..4762
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4743..4761
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4809..4829)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4809..4827)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4848..4864)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4848..4864)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4861..4883)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4872..4888
                     /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(4896..4902,4903..4920,4921..4926))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    join(4941..4945,1..17)
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccaccgg tcgtctcagg
       61 accgctagcc cgggctcgag atctgcgatc taagtaagct tgctgcttca tccccgtggc
      121 ccgttgctcg cgtttgctgg cggtgtcccc ggaagaaata tatttgcatg tctttagttc
      181 tatgatgaca caaaccccgc ccagcgtctt gtcattggcg aattcgaaca cgcagatgca
      241 gtcggggcgg cgcggtccca ggtccacttc gcatattaag gtgacgcgtg tggcctcgaa
      301 caccgagcga ccctgcagcg acccgcttaa cagcgtcaac agcgtgccgc agatcccaag
      361 cttgcggccg cttaagctgc agaagttggt cgtgaggcac tgggcaggta agtatcaagg
      421 ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag agaagactct
      481 tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt ctctccacag
      541 gtgtccactc ccaggttcaa tacagctctt aagcggccgc aagctagctt gccgccacca
      601 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
      661 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
      721 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
      781 tcgtgaccac cctgacctgg ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc
      841 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
      901 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
      961 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     1021 agctggagta caacgccatc agcgacaacg tctatatcac cgccgacaag cagaagaacg
     1081 gcatcaaggc caacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     1141 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1201 acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1261 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtgag
     1321 cctctagact ccctcagcgg cgcgccacgt tggctgtaga gtcgccaggg cgtatttaac
     1381 tggagggact ctcggttaga gttgatacgg tcccgtcgca tacagcgcct ttggccaaga
     1441 tctcacgtgt ctagagtcgg ggcggccggc cgcttcgagc agacatgata agatacattg
     1501 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     1561 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     1621 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     1681 aaaacctcta caaatgtggt aaaatcgata aggatccgtc gaccgatgcc cttgagagcc
     1741 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
     1801 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctt ccgcttcctc
     1861 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1921 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1981 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2041 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2101 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2161 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2221 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2281 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2341 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2401 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2461 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2521 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2581 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2641 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     2701 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     2761 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     2821 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     2881 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2941 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     3001 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     3061 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     3121 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     3181 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     3241 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     3301 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     3361 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     3421 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     3481 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     3541 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     3601 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     3661 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     3721 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     3781 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3841 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3901 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3961 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     4021 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4081 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4141 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4201 cgcgaatttt aacaaaatat taacgcttac aatttgccat tcgccattca ggctgcgcaa
     4261 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagccca agctaccatg
     4321 ataagtaagt aatattaagg tacgggaggt acttggagcg gccgcaataa aatatcttta
     4381 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc
     4441 catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca
     4501 ggtgccagaa catttctcta tcgataggta ccgagctcga gttccatggg agacgggcgc
     4561 cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     4621 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     4681 gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg
     4741 cgtaatacga ctcactatag ggcgaattgg gtaccgggcc ccccctcgag gtcctccagc
     4801 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     4861 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4921 tgtaaagcct ggggtgccta atgag
//
