LOCUS       sequence-433949-        4327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  KD Recombinase dependent conditional 1X ALFA cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4327)
  AUTHORS   Sanfilippo P, Kim AJ, Bhukel A, Yoo J, Mirshahidi PS, Pandey V, 
            Bevir H, Yuen A, Mirshahidi PS, Guo P, Li HS, Wohlschlegel JA, Aso 
            Y, Zipursky SL
  TITLE     Mapping of multiple neurotransmitter receptor subtypes and distinct 
            protein complexes to the connectome.
  JOURNAL   Neuron. 2024 Jan 13:S0896-6273(23)00973-X. doi: 
            10.1016/j.neuron.2023.12.014.
  PUBMED    38262414
REFERENCE   2  (bases 1 to 4327)
  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: "Neuron. 
            2024 Jan 13:S0896-6273(23)00973-X. doi: 
            10.1016/j.neuron.2023.12.014."
COMMENT     Sequence Label: pBS-KDRT-STOP-loxP-3XP3::dsRed-loxP-STOP-KDRT-...
FEATURES             Location/Qualifiers
     source          1..4327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      16..604
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     505..524
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1095..1128
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1354..1363
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1360..2037
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(1490..1508)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1540..1560)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     1758..1777
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1952..1975
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    2144..2177
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    2192..2326
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2229..2248)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2283..2302
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             2438..2476
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     primer_bind     complement(2539..2558)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2540..2558)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2568..2585)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2568..2584)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2577..2599)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2725..3180)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2862..2883)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3074..3093
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3207..3311
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3312..3380,3381..4172)
                     /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(3530..3549)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
       61 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
      121 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
      181 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
      241 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
      301 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
      361 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
      421 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
      481 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
      541 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
      601 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
      661 catgtggaaa cgatatcaga catttgtctg ataatgcttc attatcagac aaatgtctga
      721 tatcgtttta acgtaagcta gctagaccgg tgtcgactaa agccaaatag aaaattattc
      781 agttcctggc ttaagttttt aaaagtgata ttatttattt ggttgtaacc aaccaaaaga
      841 atgtaaataa ctaatacata attatgttag ttttaagtta gcaacaaatt gattttagct
      901 atattagcta cttggttaat aaatagaata tatttattta aagataattg cgtttttatt
      961 gtcagggagt gagtttgctt aaaaactcgt ttaggtttgt cctcccgaaa ttatttattt
     1021 aaatgcgatg gagagttggc gccgaatcga aaactttacg cgcttaaaag cacgagttgg
     1081 catccctaac gcgtataact tcgtataatg tatgctatac gaagttatcg tacgggatct
     1141 aattcaatta gagactaatt caattagagc taattcaatt aggatccaag cttatcgatt
     1201 tcgaaccctc gaccgccgga gtataaatag aggcgcttcg tctacggagc gacaattcaa
     1261 ttcaaacaag caaagtgaac acgtcgctaa gcgaaagcta agcaaataaa caagcgcagc
     1321 tgaacaagct aaacaatcgg ctcgaagccg gtcgccacca tggcctcctc cgaggacgtc
     1381 atcaaggagt tcatgcgctt caaggtgcgc atggagggct ccgtgaacgg ccacgagttc
     1441 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     1501 gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgtcccccca gttccagtac
     1561 ggctccaagg tgtacgtgaa gcaccccgcc gacatccccg actacaagaa gctgtccttc
     1621 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     1681 acccaggact cctccctcca ggacggctcc ttcatctaca aggtgaagtt catcggcgtg
     1741 aacttcccct ccgacggccc cgtaatgcag aagaagacta tgggctggga ggcgtccacc
     1801 gagcgcctgt acccccgcga cggcgtgctg aagggcgaga tccacaaggc cctgaagctg
     1861 aaggacggcg gccactacct ggtggagttc aagtccatct acatggccaa gaagcccgtg
     1921 cagctgcccg gctactacta cgtggactcc aagctggaca tcacctccca caacgaggac
     1981 tacaccatcg tggagcagta cgagcgcgcc gagggccgcc accacctgtt cctgtagggg
     2041 ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     2101 aacctcccac acctccccct gaacctgaaa cataaaatga atgataactt cgtataatgt
     2161 atgctatacg aagttatcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     2221 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2281 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcacta gtgatctggc
     2341 cggaaacgat atcagacatt tgtctgataa tgcttcatta tcagacaaat gtctgatatc
     2401 gtttggtggc ggcggttcgg gaggaggtgg atctccttcg cgcttggagg aggaactccg
     2461 taggaggttg actgagcccg gtggcggcgg aagcggaggt ggaggctcca catgtcaccg
     2521 cggtggagct ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     2581 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     2641 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     2701 ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta aaattcgcgt
     2761 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     2821 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     2881 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     2941 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3001 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3061 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3121 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3181 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3241 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3301 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3361 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3421 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3481 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     3541 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     3601 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     3661 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     3721 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     3781 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     3841 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     3901 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3961 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     4021 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     4081 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     4141 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     4201 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4261 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4321 tagaaaa
//
