LOCUS       sequence-258390-        4945 bp    DNA     circular SYN 15-APR-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 Apr 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_9
FEATURES             Location/Qualifiers
     source          1..4945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..146
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     intron          224..356
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     339..358
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      410..419
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(416..418,419..421,422..1132)
                     /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(461..482)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(722..741)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1069..1090
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1305..1426
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1316..1335)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1370..1389
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1674..1691)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1845..2433)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1925..1944)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2604..3395,3396..3464))
                     /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     3227..3246
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3465..3569)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3596..4051
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3683..3702)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3893..3914
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(4129..4668)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLPGN
                     SSSVPIDREMFWHLHLHWGQPILLVCFVSFCFDGERMLVLSIHTKNQHTDVMKIKIFYC
                     GRSKYLPYLNITYLSW"
     polyA_signal    4182..4230
                     /note="synthetic polyadenylation signal"
     misc_feature    4244..4335
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4284..4303
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     primer_bind     4518..4540
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4532..4549
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4533..4549
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4559..4578
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4559..4577
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4625..4645)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4625..4643)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4664..4680)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4664..4680)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4677..4699)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4688..4704
                     /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(4712..4718,4719..4736,4737..4742))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4757..4778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 atgacacaaa ccccgcccag cgtcttgtca ttggcgaatt cgaacacgca gatgcagtcg
       61 gggcggcgcg gtcccaggtc cacttcgcat attaaggtga cgcgtgtggc ctcgaacacc
      121 gagcgaccct gcagcgaccc gcttaacagc gtcaacagcg tgccgcagat cccaagcttg
      181 cggccgctta agctgcagaa gttggtcgtg aggcactggg caggtaagta tcaaggttac
      241 aagacaggtt taaggagacc aatagaaact gggcttgtcg agacagagaa gactcttgcg
      301 tttctgatag gcacctattg gtcttactga catccacttt gcctttctct ccacaggtgt
      361 ccactcccag gttcaataca gctcttaagc ggccgcaagc tagcttgccg ccaccatggt
      421 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
      481 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
      541 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
      601 gaccaccttc ggctacggcc tgatgtgctt cgcccgctac cccgaccaca tgaagcagca
      661 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
      721 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
      781 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
      841 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
      901 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
      961 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1021 gagctaccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1081 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtgagcctc
     1141 tagactccct cagcggcgcg cctgtgtcgt agttccaccg aaagacggat agctgccaga
     1201 actcgcttcg gagcatgacc acagtgaccc ggtcaaacgc cctctaaaca gtcagatctc
     1261 acgtgtctag agtcggggcg gccggccgct tcgagcagac atgataagat acattgatga
     1321 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     1381 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     1441 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     1501 cctctacaaa tgtggtaaaa tcgataagga tccgtcgacc gatgcccttg agagccttca
     1561 acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg
     1621 tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc ttcctcgctc
     1681 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     1741 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     1801 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     1861 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     1921 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     1981 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2041 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2101 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2161 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2221 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2281 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     2341 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     2401 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     2461 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     2521 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     2581 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     2641 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     2701 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     2761 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     2821 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     2881 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     2941 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3001 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3061 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3121 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3181 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3241 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3301 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     3361 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     3421 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     3481 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     3541 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg
     3601 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3661 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3721 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3781 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3841 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3901 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3961 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     4021 aattttaaca aaatattaac gcttacaatt tgccattcgc cattcaggct gcgcaactgt
     4081 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agcccaagct accatgataa
     4141 gtaagtaata ttaaggtacg ggaggtactt ggagcggccg caataaaata tctttatttt
     4201 cattacatct gtgtgttggt tttttgtgtg aatcgatagt actaacatac gctctccatc
     4261 aaaacaaaac gaaacaaaac aaactagcaa aataggctgt ccccagtgca agtgcaggtg
     4321 ccagaacatt tctctatcga taggtaccga gctcgagttg ccagggagac gggcgccgct
     4381 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     4441 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     4501 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta
     4561 atacgactca ctatagggcg aattgggtac cgggcccccc ctcgaggtcc tccagctttt
     4621 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     4681 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4741 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4801 ctttccaccg gtcgtctcct gttggctagc ccgggctcga gatctgcgat ctaagtaagc
     4861 ttgctgcttc atccccgtgg cccgttgctc gcgtttgctg gcggtgtccc cggaagaaat
     4921 atatttgcat gtctttagtt ctatg
//
