LOCUS       sequence-257556-        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_Citrine_Nanotag_11
FEATURES             Location/Qualifiers
     source          1..4945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(6..22)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6..22)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(19..41)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    30..46
                     /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(54..60,61..78,79..84))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    99..120
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        288..433
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          511..643
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     626..645
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      697..706
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(703..705,706..708,709..1419)
                     /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(748..769)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1009..1028)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1356..1377
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1592..1713
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1603..1622)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1657..1676
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1961..1978)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2132..2720)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2212..2231)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2891..3682,3683..3751))
                     /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     3514..3533
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3752..3856)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3883..4338
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3970..3989)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4180..4201
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4416..4945,1..10))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4469..4517
                     /note="synthetic polyadenylation signal"
     misc_feature    4531..4622
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4571..4590
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4805..4827
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4819..4836
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4820..4836
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4846..4865
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4846..4864
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4912..4932)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4912..4930)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
       61 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
      121 attgcgttgc gctcactgcc cgctttccac cggtcgtctc ctgttggcta gcccgggctc
      181 gagatctgcg atctaagtaa gcttgctgct tcatccccgt ggcccgttgc tcgcgtttgc
      241 tggcggtgtc cccggaagaa atatatttgc atgtctttag ttctatgatg acacaaaccc
      301 cgcccagcgt cttgtcattg gcgaattcga acacgcagat gcagtcgggg cggcgcggtc
      361 ccaggtccac ttcgcatatt aaggtgacgc gtgtggcctc gaacaccgag cgaccctgca
      421 gcgacccgct taacagcgtc aacagcgtgc cgcagatccc aagcttgcgg ccgcttaagc
      481 tgcagaagtt ggtcgtgagg cactgggcag gtaagtatca aggttacaag acaggtttaa
      541 ggagaccaat agaaactggg cttgtcgaga cagagaagac tcttgcgttt ctgataggca
      601 cctattggtc ttactgacat ccactttgcc tttctctcca caggtgtcca ctcccaggtt
      661 caatacagct cttaagcggc cgcaagctag cttgccgcca ccatggtgag caagggcgag
      721 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
      781 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
      841 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccttcggc
      901 tacggcctga tgtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
      961 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     1021 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     1081 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     1141 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     1201 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     1261 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag ctaccagtcc
     1321 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     1381 gccgccggga tcactctcgg catggacgag ctgtacaagt gagcctctag actccctcag
     1441 cggcgcgcct actcgtccgg cgtcacaaat tagcggaccg accactgcac ctctagcgta
     1501 ccgtcaaagg gcaacgaggg tcattccgtg ccgacataag agatctcacg tgtctagagt
     1561 cggggcggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc
     1621 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     1681 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     1741 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     1801 ggtaaaatcg ataaggatcc gtcgaccgat gcccttgaga gccttcaacc cagtcagctc
     1861 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     1921 gcaactcgta ggacaggtgc cggcagcgct cttccgcttc ctcgctcact gactcgctgc
     1981 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2041 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2101 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2161 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2221 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2281 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2341 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2401 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2461 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2521 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2581 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2641 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2701 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2761 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     2821 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     2881 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     2941 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3001 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3061 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3121 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3181 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3241 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3301 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3361 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3421 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3481 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3541 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3601 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3661 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3721 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     3781 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3841 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg
     3901 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3961 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4021 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4081 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4141 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     4201 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     4261 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     4321 tattaacgct tacaatttgc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     4381 cggtgcgggc ctcttcgcta ttacgccagc ccaagctacc atgataagta agtaatatta
     4441 aggtacggga ggtacttgga gcggccgcaa taaaatatct ttattttcat tacatctgtg
     4501 tgttggtttt ttgtgtgaat cgatagtact aacatacgct ctccatcaaa acaaaacgaa
     4561 acaaaacaaa ctagcaaaat aggctgtccc cagtgcaagt gcaggtgcca gaacatttct
     4621 ctatcgatag gtaccgagct cgagttgcca gggagacggg cgccgctaca gggcgcgtcc
     4681 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     4741 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
     4801 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta
     4861 tagggcgaat tgggtaccgg gccccccctc gaggtcctcc agcttttgtt ccctttagtg
     4921 agggttaatt gcgcgcttgg cgtaa
//
