LOCUS       sequence-257560-        4951 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 4951)
  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 4951)
  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_35
FEATURES             Location/Qualifiers
     source          1..4951
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     67..89
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     81..98
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     82..98
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     108..127
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        108..126
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(174..194)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(174..192)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(213..229)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(213..229)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(226..248)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    237..253
                     /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(261..267,268..285,286..291))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    306..327
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        495..640
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          718..850
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     833..852
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      904..913
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(910..912,913..915,916..1626)
                     /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(955..976)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1216..1235)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1563..1584
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1805..1926
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1816..1835)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1870..1889
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2174..2191)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2345..2933)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2425..2444)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3104..3895,3896..3964))
                     /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     3727..3746
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3965..4069)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4096..4551
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4183..4202)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4393..4414
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4629..4951,1..217))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4682..4730
                     /note="synthetic polyadenylation signal"
     misc_feature    4744..4835
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4784..4803
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
       61 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac
      121 tatagggcga attgggtacc gggccccccc tcgaggtcct ccagcttttg ttccctttag
      181 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
      241 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
      301 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccaccgg
      361 tcgtctcctg ttggctagcc cgggctcgag atctgcgatc taagtaagct tgctgcttca
      421 tccccgtggc ccgttgctcg cgtttgctgg cggtgtcccc ggaagaaata tatttgcatg
      481 tctttagttc tatgatgaca caaaccccgc ccagcgtctt gtcattggcg aattcgaaca
      541 cgcagatgca gtcggggcgg cgcggtccca ggtccacttc gcatattaag gtgacgcgtg
      601 tggcctcgaa caccgagcga ccctgcagcg acccgcttaa cagcgtcaac agcgtgccgc
      661 agatcccaag cttgcggccg cttaagctgc agaagttggt cgtgaggcac tgggcaggta
      721 agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag
      781 agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt
      841 ctctccacag gtgtccactc ccaggttcaa tacagctctt aagcggccgc aagctagctt
      901 gccgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
      961 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     1021 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1081 tggcccaccc tcgtgaccac cttcggctac ggcctgatgt gcttcgcccg ctaccccgac
     1141 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     1201 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1261 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1321 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     1381 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     1441 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1501 gacaaccact acctgagcta ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     1561 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     1621 tacaagtgag cctctagact ccctcagcgg cgcgccgatg cgaaggagtt tgcgtaccac
     1681 gaaccttgct tgagcacagg tccgaggctc aactaagggt ttgggacggg ccaacgtagc
     1741 tgactgctat gccagatctc acgtgtctag agtcggggcg gccggccgct tcgagcagac
     1801 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     1861 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     1921 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     1981 gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tccgtcgacc
     2041 gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat
     2101 cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc
     2161 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2221 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     2281 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     2341 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     2401 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     2461 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     2521 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     2581 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     2641 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2701 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     2761 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     2821 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     2881 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     2941 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3001 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3061 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3121 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3181 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3241 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3301 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     3361 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     3421 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     3481 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     3541 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     3601 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3661 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3721 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     3781 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     3841 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     3901 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     3961 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4021 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4081 gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4141 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4201 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4261 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4321 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4381 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     4441 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     4501 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tgccattcgc
     4561 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     4621 agcccaagct accatgataa gtaagtaata ttaaggtacg ggaggtactt ggagcggccg
     4681 caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagt
     4741 actaacatac gctctccatc aaaacaaaac gaaacaaaac aaactagcaa aataggctgt
     4801 ccccagtgca agtgcaggtg ccagaacatt tctctatcga taggtaccga gctcgagttg
     4861 ccagggagac gggcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
     4921 tgggaagggc gatcggtgcg ggcctcttcg c
//
