LOCUS       sequence-258988-        4944 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 4944)
  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 4944)
  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_45
FEATURES             Location/Qualifiers
     source          1..4944
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..19
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3..19
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     29..48
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        29..47
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(95..115)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(95..113)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(134..150)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(134..150)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(147..169)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    158..174
                     /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(182..188,189..206,207..212))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    227..248
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        416..561
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          639..771
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     754..773
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      825..834
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(831..833,834..836,837..1547)
                     /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(876..897)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1137..1156)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1484..1505
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1719..1840
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1730..1749)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1784..1803
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2088..2105)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2259..2847)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2339..2358)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3018..3809,3810..3878))
                     /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     3641..3660
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3879..3983)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4010..4465
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4097..4116)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4307..4328
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4543..4944,1..138))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4596..4644
                     /note="synthetic polyadenylation signal"
     misc_feature    4658..4749
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4698..4717
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     join(4932..4944,1..10)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
       61 cgggcccccc ctcgaggtcc tccagctttt gttcccttta gtgagggtta attgcgcgct
      121 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
      181 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
      241 tcacattaat tgcgttgcgc tcactgcccg ctttccaccg gtcgtctcct gttggctagc
      301 ccgggctcga gatctgcgat ctaagtaagc ttgctgcttc atccccgtgg cccgttgctc
      361 gcgtttgctg gcggtgtccc cggaagaaat atatttgcat gtctttagtt ctatgatgac
      421 acaaaccccg cccagcgtct tgtcattggc gaattcgaac acgcagatgc agtcggggcg
      481 gcgcggtccc aggtccactt cgcatattaa ggtgacgcgt gtggcctcga acaccgagcg
      541 accctgcagc gacccgctta acagcgtcaa cagcgtgccg cagatcccaa gcttgcggcc
      601 gcttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
      661 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
      721 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
      781 cccaggttca atacagctct taagcggccg caagctagct tgccgccacc atggtgagca
      841 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
      901 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
      961 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1021 ccttcggcta cggcctgatg tgcttcgccc gctaccccga ccacatgaag cagcacgact
     1081 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     1141 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1201 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1261 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     1321 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     1381 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct
     1441 accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1501 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga gcctctagac
     1561 tccctcagcg gcgcgcctgg tgttcctcga gatcggagtc ctaaatcgac tgccttagcg
     1621 gtaggacagt agatcagtca gcgacgtgac gaaccctcgc aggatcaaga tcccacgtgt
     1681 ctagagtcgg ggcggccggc cgcttcgagc agacatgata agatacattg atgagtttgg
     1741 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     1801 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     1861 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     1921 caaatgtggt aaaatcgata aggatccgtc gaccgatgcc cttgagagcc ttcaacccag
     1981 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct
     2041 ttatcatgca actcgtagga caggtgccgg cagcgctctt ccgcttcctc gctcactgac
     2101 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     2161 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     2221 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     2281 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     2341 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     2401 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     2461 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     2521 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     2581 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     2641 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     2701 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2761 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2821 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2881 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     2941 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3001 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3061 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3121 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3181 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3241 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3301 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3361 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3421 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3481 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3541 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3601 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3661 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3721 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3781 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3841 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     3901 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     3961 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgcgccctgt
     4021 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4081 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4141 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4201 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     4261 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4321 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     4381 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     4441 aacaaaatat taacgcttac aatttgccat tcgccattca ggctgcgcaa ctgttgggaa
     4501 gggcgatcgg tgcgggcctc ttcgctatta cgccagccca agctaccatg ataagtaagt
     4561 aatattaagg tacgggaggt acttggagcg gccgcaataa aatatcttta ttttcattac
     4621 atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc catcaaaaca
     4681 aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca ggtgccagaa
     4741 catttctcta tcgataggta ccgagctcga gttgccaggg agacgggcgc cgctacaggg
     4801 cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     4861 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     4921 gccagggttt tcccagtcac gacg
//
