LOCUS       sequence-257559-        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_Citrine_Nanotag_18
FEATURES             Location/Qualifiers
     source          1..4947
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          78..210
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     193..212
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      264..273
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(270..272,273..275,276..986)
                     /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(315..336)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(576..595)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     923..944
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1161..1282
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1172..1191)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1226..1245
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1530..1547)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1701..2289)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1781..1800)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2460..3251,3252..3320))
                     /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     3083..3102
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3321..3425)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3452..3907
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3539..3558)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3749..3770
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3985..4524)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4038..4086
                     /note="synthetic polyadenylation signal"
     misc_feature    4100..4191
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4140..4159
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4374..4396
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4388..4405
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4389..4405
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4415..4434
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4415..4433
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4481..4501)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4481..4499)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4520..4536)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4520..4536)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4533..4555)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4544..4560
                     /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(4568..4574,4575..4592,4593..4598))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4613..4634
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4802..4947
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
ORIGIN
        1 cagcgtcaac agcgtgccgc agatcccaag cttgcggccg cttaagctgc agaagttggt
       61 cgtgaggcac tgggcaggta agtatcaagg ttacaagaca ggtttaagga gaccaataga
      121 aactgggctt gtcgagacag agaagactct tgcgtttctg ataggcacct attggtctta
      181 ctgacatcca ctttgccttt ctctccacag gtgtccactc ccaggttcaa tacagctctt
      241 aagcggccgc aagctagctt gccgccacca tggtgagcaa gggcgaggag ctgttcaccg
      301 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
      361 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
      421 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgatgt
      481 gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
      541 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
      601 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
      661 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
      721 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
      781 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
      841 acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccgcc ctgagcaaag
      901 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
      961 ctctcggcat ggacgagctg tacaagtgag cctctagact ccctcagcgg cgcgccttgc
     1021 agtaattcac gaggccaaag gcgaaccatt tccaccagcg gtactccggc tcattcttac
     1081 cgcgcagatc gttgtggaca gatcgcgaaa gatctcacgt gtctagagtc ggggcggccg
     1141 gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     1201 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     1261 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     1321 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga
     1381 taaggatccg tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     1441 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     1501 gacaggtgcc ggcagcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     1561 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     1621 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     1681 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     1741 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1801 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     1861 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     1921 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     1981 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2041 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     2101 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     2161 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     2221 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     2281 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     2341 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     2401 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     2461 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     2521 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     2581 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     2641 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     2701 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     2761 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     2821 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     2881 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     2941 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3001 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3061 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3121 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3181 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     3241 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     3301 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     3361 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     3421 ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg
     3481 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     3541 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     3601 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     3661 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     3721 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     3781 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     3841 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     3901 acaatttgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
     3961 tcttcgctat tacgccagcc caagctacca tgataagtaa gtaatattaa ggtacgggag
     4021 gtacttggag cggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt
     4081 tgtgtgaatc gatagtacta acatacgctc tccatcaaaa caaaacgaaa caaaacaaac
     4141 tagcaaaata ggctgtcccc agtgcaagtg caggtgccag aacatttctc tatcgatagg
     4201 taccgagctc gagttgccag ggagacgggc gccgctacag ggcgcgtccc attcgccatt
     4261 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     4321 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     4381 acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt
     4441 gggtaccggg ccccccctcg aggtcctcca gcttttgttc cctttagtga gggttaattg
     4501 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4561 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4621 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccaccggtcg tctcctgttg
     4681 gctagcccgg gctcgagatc tgcgatctaa gtaagcttgc tgcttcatcc ccgtggcccg
     4741 ttgctcgcgt ttgctggcgg tgtccccgga agaaatatat ttgcatgtct ttagttctat
     4801 gatgacacaa accccgccca gcgtcttgtc attggcgaat tcgaacacgc agatgcagtc
     4861 ggggcggcgc ggtcccaggt ccacttcgca tattaaggtg acgcgtgtgg cctcgaacac
     4921 cgagcgaccc tgcagcgacc cgcttaa
//
