LOCUS       sequence-257577-        4947 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 4947)
  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 4947)
  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_62
FEATURES             Location/Qualifiers
     source          1..4947
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        190..335
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          413..545
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     528..547
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      599..608
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(605..607,608..610,611..1324)
                     /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(650..671)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(911..930)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1258..1279
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1496..1617
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1507..1526)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1561..1580
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1865..1882)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2036..2624)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2116..2135)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2795..3586,3587..3655))
                     /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     3418..3437
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3656..3760)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3787..4242
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3874..3893)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4084..4105
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4320..4859)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPWN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4373..4421
                     /note="synthetic polyadenylation signal"
     misc_feature    4435..4526
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4475..4494
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4709..4731
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4723..4740
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4724..4740
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4750..4769
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4750..4768
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4816..4836)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4816..4834)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4855..4871)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4855..4871)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4868..4890)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4879..4895
                     /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(4903..4909,4910..4927,4928..4933))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc accggtcgtc
       61 tcaggaccgc tagcccgggc tcgagatctg cgatctaagt aagcttgctg cttcatcccc
      121 gtggcccgtt gctcgcgttt gctggcggtg tccccggaag aaatatattt gcatgtcttt
      181 agttctatga tgacacaaac cccgcccagc gtcttgtcat tggcgaattc gaacacgcag
      241 atgcagtcgg ggcggcgcgg tcccaggtcc acttcgcata ttaaggtgac gcgtgtggcc
      301 tcgaacaccg agcgaccctg cagcgacccg cttaacagcg tcaacagcgt gccgcagatc
      361 ccaagcttgc ggccgcttaa gctgcagaag ttggtcgtga ggcactgggc aggtaagtat
      421 caaggttaca agacaggttt aaggagacca atagaaactg ggcttgtcga gacagagaag
      481 actcttgcgt ttctgatagg cacctattgg tcttactgac atccactttg cctttctctc
      541 cacaggtgtc cactcccagg ttcaatacag ctcttaagcg gccgcaagct agcttgccgc
      601 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      661 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      721 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      781 caccctcgtg accaccctga cctggggcgt gcagtgcttc gcccgctacc ccgaccacat
      841 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      901 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
      961 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1021 gcacaagctg gagtacaacg ccatcagcga caacgtctat atcaccgccg acaagcagaa
     1081 gaacggcatc aaggccaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1141 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1201 ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat
     1261 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1321 gtgagcctct agactccctc agcggcgcgc cacttttcgg ttctggtgga cgtaatcaat
     1381 cagcgaacgc cgtcttctgc cgtcttctgg taacgcacga actctgccga gcaacctaca
     1441 gaccagatct cacgtgtcta gagtcggggc ggccggccgc ttcgagcaga catgataaga
     1501 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     1561 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     1621 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     1681 agcaagtaaa acctctacaa atgtggtaaa atcgataagg atccgtcgac cgatgccctt
     1741 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     1801 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctcttccg
     1861 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     1921 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     1981 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     2041 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     2101 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     2161 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     2221 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     2281 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     2341 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     2401 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     2461 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     2521 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     2581 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     2641 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     2701 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     2761 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     2821 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     2881 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     2941 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     3001 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     3061 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     3121 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     3181 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     3241 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     3301 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     3361 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     3421 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     3481 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     3541 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     3601 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     3661 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     3721 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     3781 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3841 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     3901 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3961 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4021 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4081 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4141 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4201 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttgccattcg ccattcaggc
     4261 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagcccaagc
     4321 taccatgata agtaagtaat attaaggtac gggaggtact tggagcggcc gcaataaaat
     4381 atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag tactaacata
     4441 cgctctccat caaaacaaaa cgaaacaaaa caaactagca aaataggctg tccccagtgc
     4501 aagtgcaggt gccagaacat ttctctatcg ataggtaccg agctcgagtt ccatgggaga
     4561 cgggcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg
     4621 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     4681 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     4741 gagcgcgcgt aatacgactc actatagggc gaattgggta ccgggccccc cctcgaggtc
     4801 ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata
     4861 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4921 cataaagtgt aaagcctggg gtgccta
//
