LOCUS       sequence-366114-        4379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to synthesize RNA probe for in situ hybridization for nmbb.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4379)
  AUTHORS   Spikol ED, Cheng J, Macurak M, Subedi A, Halpern ME
  TITLE     Genetically defined nucleus incertus neurons differ in connectivity 
            and function.
  JOURNAL   Elife. 2024 May 31;12:RP89516. doi: 10.7554/eLife.89516.
  PUBMED    38819436
REFERENCE   2  (bases 1 to 4379)
  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"; journalName: "Elife."; 
            date: "2024-05-31"; volume: "12:RP89516. doi"; pages: " 
            10.7554/eLife.89516"
COMMENT     Sequence Label: TOPO-nmbb
FEATURES             Location/Qualifiers
     source          1..4379
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        239..257
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     239..256
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(812..831)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(813..831)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(838..855)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(838..854)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(847..869)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      995..1423
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1082..1101)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1292..1313
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             1767..2561
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1821..1840)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2431..2450
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(2579..2647,2648..3439)
                     /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     complement(2797..2816)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3610..4198
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4099..4118
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4352..4369
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat
      241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca
      301 ctagtaacgg ccgccagtgt gctggaattc gcccttcagt ccaagcgtat ccaggttttg
      361 ttgctgtgga ttcctcacat acctagtgct tttctctttt tacatttcga aaacgtcgtc
      421 agtgagcctc gacttgattg aacttagaaa caaagttgca aagatcaaag taaaccccag
      481 aggaaattta tgggcaacag gtcattttat ggggaagaaa agtgtcgtgg acagcaagca
      541 cttgccgacc gaggatgagt ccacgatgac agccgtcgag gctgcgctga acgcccggca
      601 ggtcgagccc gaggatgttt tccaggagat gctgagagtc gctctgcaaa cacacctcga
      661 cacgcgccac atccgcccga acgtcccgga gactgcaata ttaatgaagg tattagaaag
      721 ctacattcaa gacaataaat gaaagggcga attctgcaga tatccatcac actggcggcc
      781 gctcgagcat gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact
      841 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
      901 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
      961 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc
     1021 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1081 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1141 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1201 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1261 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1321 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1381 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aattcagggc
     1441 gcaagggctg ctaaaggaag cggaacacgt agaaagccag tccgcagaaa cggtgctgac
     1501 cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc gcaaagagaa
     1561 agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt ttatggacag
     1621 caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag
     1681 taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca agatctgatc
     1741 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     1801 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     1861 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     1921 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     1981 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     2041 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc ccaccttgct cctgccgaga
     2101 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     2161 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     2221 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     2281 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     2341 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     2401 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     2461 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     2521 agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattgaaaaa ggaagagtat
     2581 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     2641 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     2701 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     2761 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     2821 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     2881 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     2941 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     3001 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     3061 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     3121 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     3181 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     3241 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     3301 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3361 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3421 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     3481 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3541 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3601 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3661 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3721 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     3781 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3841 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3901 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3961 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     4021 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     4081 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     4141 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4201 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4261 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4321 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaag
//
