LOCUS       sequence-366115-        4403 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Used to synthesize RNA probe for in situ hybridization for nts.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4403)
  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 4403)
  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"; journalName: "Elife."; 
            date: "2024-05-31"; volume: "12:RP89516. doi"; pages: " 
            10.7554/eLife.89516"
COMMENT     Sequence Label: TOPO-nts
FEATURES             Location/Qualifiers
     source          1..4403
                     /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(836..855)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(837..855)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(862..879)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(862..878)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(871..893)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1019..1447
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1106..1125)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1316..1337
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             1791..2585
                     /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(1845..1864)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2455..2474
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(2603..2671,2672..3463)
                     /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(2821..2840)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3634..4222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4123..4142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4376..4393
                     /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 gcccttttgt gtgttttctc cctcttcaga
      361 tatagaccag ggaaaaagag caatagagga tgaggttctg agaagtctcc tcacatcaaa
      421 ggtaaaggcg agcaggcaca tcgcccccct gtggcagctt cctctgcagg acgtgtgcag
      481 gatggtgaac gggctcggtg actcgtggct ggaagcctgg gccaatgagg aggcggcgga
      541 ggacactgag gtccacgctg actatgagca gagggtctcg gggactcttc ttcagatgct
      601 ggaggaaatg catgacattc agaacctctg cagggtcctg caacctagag agcttcaaga
      661 cgagcaggaa tatctggagc tggagcaaaa cagcgacagt ccgctgaaga ggaaatctcc
      721 ctatatactg aaaagacaac tgcgcactaa taaatccaga cggccgaagg gcgaattctg
      781 cagatatcca tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta
      841 tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
      901 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
      961 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac
     1021 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1081 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1141 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1201 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1261 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1321 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     1381 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1441 gcgaatttta acaaaattca gggcgcaagg gctgctaaag gaagcggaac acgtagaaag
     1501 ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa
     1561 gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca tggcgatagc
     1621 tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg gcgccctctg
     1681 gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca aggatctgat
     1741 ggcgcagggg atcaagatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     1801 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     1861 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     1921 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     1981 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     2041 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     2101 catcccacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     2161 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     2221 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     2281 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     2341 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     2401 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     2461 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     2521 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     2581 tctgaattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     2641 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     2701 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     2761 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     2821 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     2881 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     2941 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3001 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3061 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3121 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     3181 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3241 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     3301 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     3361 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     3421 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     3481 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     3541 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     3601 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3661 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3721 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3781 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3841 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3901 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3961 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4021 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4081 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4141 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4201 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4261 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     4321 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     4381 gtcagtgagc gaggaagcgg aag
//
