LOCUS       sequence-494695-        5835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The set of RNA constructs used to produce barcode library in the 
            optimized connectome-seq experiments. Also used for Basescope 
            experiment. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5835)
  AUTHORS   Chen D, Isakova A, Wan ZJ, Wagner MJ, Wu Y, Zhao BS
  TITLE     Connectome-seq: High-throughput Mapping of Neuronal Connectivity at 
            Single-Synapse Resolution via Barcode Sequencing.
  JOURNAL   bioRxiv [Preprint]. 2025 May 26:2025.02.13.638129. doi: 
            10.1101/2025.02.13.638129.
  PUBMED    41030982
REFERENCE   2  (bases 1 to 5835)
  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: "bioRxiv 
            [Preprint]. 2025 May 26:2025.02.13.638129. doi: 
            10.1101/2025.02.13.638129."
COMMENT     Sequence Label: hSyn-Cre-hSyn-PreRNA barcode landing pad (PPIB)
FEATURES             Location/Qualifiers
     source          1..5835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        181..628
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             642..1673
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     primer_bind     complement(1734..1754)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     promoter        2136..2583
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    2851..2869
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     protein_bind    2875..2893
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
     polyA_signal    2937..3058
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3028..3047
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3098..3238
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3098..3227
                     /label=AAV2 ITR
     rep_origin      3313..3768
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3400..3419)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3610..3631
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3785..3804)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3904..3926
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3964..3982)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4050..4154
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4155..4223,4224..5015)
                     /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(4373..4392)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5186..5774
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5675..5694
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgttgtaca gtgtctagac tgcagagggc cctgcgtatg
      181 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga
      241 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga
      301 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg
      361 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc
      421 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg
      481 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg
      541 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg
      601 aggagtcgtg tcgtgcctga gagcgcagtc gagaaggatc catgtccaat ttactgaccg
      661 tacaccaaaa tttgcctgca ttaccggtcg atgcaacgag tgatgaggtt cgcaagaacc
      721 tgatggacat gttcagggat cgccaggcgt tttctgagca tacctggaaa atgcttctgt
      781 ccgtttgccg gtcgtgggcg gcatggtgca agttgaataa ccggaaatgg tttcccgcag
      841 aacctgaaga tgttcgcgat tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa
      901 ctatccagca acatttgggc cagctaaaca tgcttcatcg tcggtccggg ctgccacgac
      961 caagtgacag caatgctgtt tcactggtta tgcggcggat ccgaaaagaa aacgttgatg
     1021 ccggtgaacg tgcaaaacag gctctagcgt tcgaacgcac tgatttcgac caggttcgtt
     1081 cactcatgga aaatagcgat cgctgccagg atatacgtaa tctggcattt ctggggattg
     1141 cttataacac cctgttacgt atagccgaaa ttgccaggat cagggttaaa gatatctcac
     1201 gtactgacgg tgggagaatg ttaatccata ttggcagaac gaaaacgctg gttagcaccg
     1261 caggtgtaga gaaggcactt agcctggggg taactaaact ggtcgagcga tggatttccg
     1321 tctctggtgt agctgatgat ccgaataact acctgttttg ccgggtcaga aaaaatggtg
     1381 ttgccgcgcc atctgccacc agccagctat caactcgcgc cctggaaggg atttttgaag
     1441 caactcatcg attgatttac ggcgctaagg atgactctgg tcagagatac ctggcctggt
     1501 ctggacacag tgcccgtgtc ggagccgcgc gagatatggc ccgcgctgga gtttcaatac
     1561 cggagatcat gcaagctggt ggctggacca atgtaaatat tgtcatgaac tatatccgta
     1621 acctggatag tgaaacaggg gcaatggtgc gcctgctgga agatggcgat tagactagta
     1681 taatcaacct ctggattaca aaattgtgaa agattgactg gtattcttaa ctatgttgct
     1741 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     1801 atggctttca ttttctcctc cttgtataaa tcctggttag ttcttgccac ggcggaactc
     1861 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     1921 gtggtgttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat ctagctttat
     1981 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     2041 taacaacaac attgcattca ttttatgttt caggttcagg gggagatgtg ggagcatgct
     2101 ggggagtgtc tagactgcag agggccctgc gtatgagtgc aagtgggttt taggaccagg
     2161 atgaggcggg gtgggggtgc ctacctgacg accgaccccg acccactgga caagcaccca
     2221 acccccattc cccaaattgc gtatccccta tcagagaggg ggaggggaaa caggatgcgg
     2281 cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag tgccttcgcc cccgcctggc
     2341 ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga cgtcactcgc cggtcccccg
     2401 caaactcccc ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc agccggaccg
     2461 caccacgcga ggcgcgagat aggggggcac gggcgcgacc atctgcgctg cggcgccggc
     2521 gactcagcgc tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg cctgagagcg
     2581 cagcaattgg agaccggaga agccctttgc catcgccaag gagtagggca cagggacatc
     2641 tttctttgag tgaccgtctg tgcaggccct gtagtccgcc acagggctct gagctgcact
     2701 ggccccggtg ctggcatctg gtggagcgga cccactcccc tcacattcca caggcccatg
     2761 gactcacttt ggtctcatgc atcctctttt tccggctgga accttcccta agtccaacta
     2821 ctaaactggg ctaagtccaa ctactaaact gggccctgaa gaagggccca tatagggccc
     2881 tgaagaaggg ccctatcgag gatattatct cgaggcttta aggccggtcc tagcaaaact
     2941 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3001 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaca
     3061 tgctggggag gtaaccacgt gcggaccgag cggccgcagg aacccctagt gatggagttg
     3121 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     3181 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     3241 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     3301 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3361 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     3421 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3481 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     3541 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3601 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     3661 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     3721 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     3781 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     3841 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3901 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     3961 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     4021 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     4081 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     4141 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     4201 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     4261 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     4321 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     4381 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     4441 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     4501 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     4561 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     4621 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     4681 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     4741 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     4801 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     4861 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     4921 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     4981 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     5041 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     5101 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     5161 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     5221 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     5281 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     5341 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     5401 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     5461 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     5521 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5581 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5641 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5701 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5761 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5821 cttttgctca catgt
//
