LOCUS       sequence-366113-        4366 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to synthesize RNA probe for in situ hybridization for nmba.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4366)
  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 4366)
  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-nmba
FEATURES             Location/Qualifiers
     source          1..4366
                     /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"
     CDS             217..1005
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSYLGDTIEYSSYASSLVPSSDPLVTAASVLEFALMADDG
                     HCKYTLLFALIMLFNVSLCTSVSLDLTELRNKVSKIKVHPRGNLWATGHFMGKKSISNS
                     QLQDSPFPVKPDRNIMGESGSSEDLKELITQEMLKIALQAQLEDPKRTRDVYNQVIREI
                     FKELANRMKGEFCRYPSHWRPLEHASRGPNSPYSESYYNSLAVVLQRRDWENPGVTQLN
                     RLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWTRPVAAH"
     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(799..818)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(800..818)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(825..842)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(825..841)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(834..856)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      982..1410
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1069..1088)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1279..1300
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             1754..2548
                     /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(1808..1827)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2418..2437
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(2566..2634,2635..3426)
                     /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(2784..2803)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3597..4185
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4086..4105
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4339..4356
                     /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 gcccttatgg ctgatgatgg acattgcaaa
      361 tatacattgt tattcgcgct cataatgctt ttcaacgtat cactgtgcac ttcagtcagt
      421 ctcgatttaa cggagctgcg aaataaagtt tcgaaaataa aagtgcatcc acgtggaaat
      481 ctttgggcaa caggtcattt tatgggcaaa aagagcattt caaacagcca gctccaggat
      541 tccccgttcc ccgtaaaacc agataggaat attatgggag aatcagggag ttcagaggat
      601 ttgaaggagc tcatcactca ggagatgctg aaaattgctc ttcaggctca gcttgaagat
      661 ccaaaaagga cacgagatgt ttataatcag gtgattaggg agatatttaa agaattggcc
      721 aacaggatga agggcgaatt ctgcagatat ccatcacact ggcggccgct cgagcatgca
      781 tctagagggc ccaattcgcc ctatagtgag tcgtattaca attcactggc cgtcgtttta
      841 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
      901 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
      961 cgcagcctga atggcgaatg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1021 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1081 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1141 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1201 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1261 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1321 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1381 agctgattta acaaaaattt aacgcgaatt ttaacaaaat tcagggcgca agggctgcta
     1441 aaggaagcgg aacacgtaga aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt
     1501 cagctactgg gctatctgga caagggaaaa cgcaagcgca aagagaaagc aggtagcttg
     1561 cagtgggctt acatggcgat agctagactg ggcggtttta tggacagcaa gcgaaccgga
     1621 attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc
     1681 tttcttgccg ccaaggatct gatggcgcag gggatcaaga tctgatcaag agacaggatg
     1741 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     1801 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     1861 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     1921 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     1981 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     2041 agtgccgggg caggatctcc tgtcatccca ccttgctcct gccgagaaag tatccatcat
     2101 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     2161 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     2221 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     2281 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     2341 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     2401 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     2461 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     2521 ctatcgcctt cttgacgagt tcttctgaat tgaaaaagga agagtatgag tattcaacat
     2581 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     2641 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     2701 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     2761 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     2821 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     2881 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     2941 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3001 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3061 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     3121 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3181 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3241 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     3301 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3361 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3421 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     3481 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     3541 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3601 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3661 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3721 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     3781 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3841 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3901 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3961 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4021 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4081 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4141 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4201 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4261 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4321 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaag
//
