LOCUS       sequence-257578-        4942 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 4942)
  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 4942)
  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_66
FEATURES             Location/Qualifiers
     source          1..4942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    15..36
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        204..349
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          427..559
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     542..561
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      613..622
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(619..621,622..624,625..1338)
                     /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(664..685)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(925..944)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1272..1293
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1505..1626
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1516..1535)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1570..1589
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1874..1891)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2045..2633)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2125..2144)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2804..3595,3596..3664))
                     /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     3427..3446
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3665..3769)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3796..4251
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3883..3902)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4093..4114
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4329..4868)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPWN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4382..4430
                     /note="synthetic polyadenylation signal"
     misc_feature    4444..4535
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4484..4503
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4718..4740
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4732..4749
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4733..4749
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4759..4778
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4759..4777
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4825..4845)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4825..4843)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4864..4880)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4864..4880)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4877..4899)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4888..4904
                     /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(4912..4918,4919..4936,4937..4942))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
       61 ttccaccggt cgtctcagga ccgctagccc gggctcgaga tctgcgatct aagtaagctt
      121 gctgcttcat ccccgtggcc cgttgctcgc gtttgctggc ggtgtccccg gaagaaatat
      181 atttgcatgt ctttagttct atgatgacac aaaccccgcc cagcgtcttg tcattggcga
      241 attcgaacac gcagatgcag tcggggcggc gcggtcccag gtccacttcg catattaagg
      301 tgacgcgtgt ggcctcgaac accgagcgac cctgcagcga cccgcttaac agcgtcaaca
      361 gcgtgccgca gatcccaagc ttgcggccgc ttaagctgca gaagttggtc gtgaggcact
      421 gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg
      481 tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac tgacatccac
      541 tttgcctttc tctccacagg tgtccactcc caggttcaat acagctctta agcggccgca
      601 agctagcttg ccgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      661 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      721 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      781 cccgtgccct ggcccaccct cgtgaccacc ctgacctggg gcgtgcagtg cttcgcccgc
      841 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      901 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
      961 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1021 ggcaacatcc tggggcacaa gctggagtac aacgccatca gcgacaacgt ctatatcacc
     1081 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac
     1141 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1201 ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag
     1261 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1321 gacgagctgt acaagtgagc ctctagactc cctcagcggc gcgccactgg tacgagaacg
     1381 gtggagtacc tttcgagcgc cggtaagaag cgtcgcctga ctaaaggttc gggtgcagac
     1441 cagttccgac acgcgatctc acgtgtctag agtcggggcg gccggccgct tcgagcagac
     1501 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     1561 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     1621 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     1681 gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tccgtcgacc
     1741 gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat
     1801 cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc
     1861 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     1921 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     1981 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2041 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     2101 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     2161 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2221 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2281 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2341 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2401 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     2461 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     2521 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     2581 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     2641 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     2701 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     2761 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     2821 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     2881 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     2941 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3001 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     3061 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     3121 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3181 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3241 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3301 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3361 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3421 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     3481 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     3541 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     3601 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     3661 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     3721 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     3781 gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3841 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3901 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3961 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4021 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4081 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4141 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4201 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tgccattcgc
     4261 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     4321 agcccaagct accatgataa gtaagtaata ttaaggtacg ggaggtactt ggagcggccg
     4381 caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagt
     4441 actaacatac gctctccatc aaaacaaaac gaaacaaaac aaactagcaa aataggctgt
     4501 ccccagtgca agtgcaggtg ccagaacatt tctctatcga taggtaccga gctcgagttc
     4561 catgggagac gggcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
     4621 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     4681 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     4741 acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac cgggcccccc
     4801 ctcgaggtcc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     4861 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4921 agccggaagc ataaagtgta aa
//
