LOCUS       sequence-494696-        5866 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 5866)
  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 5866)
  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-PostRNA barcode landing pad (dapB)
FEATURES             Location/Qualifiers
     source          1..5866
                     /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    2783..2801
                     /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    2807..2825
                     /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)"
     primer_bind     complement(2887..2904)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2893..3100
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3129..3269
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3129..3258
                     /label=AAV2 ITR
     rep_origin      3344..3799
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3431..3450)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3641..3662
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3816..3835)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3935..3957
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3995..4013)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4081..4185
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4186..4254,4255..5046)
                     /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(4404..4423)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5217..5805
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5706..5725
                     /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 cgcgagtgtc tagactgcag agggccctgc gtatgagtgc aagtgggttt taggaccagg
     2161 atgaggcggg gtgggggtgc ctacctgacg accgaccccg acccactgga caagcaccca
     2221 acccccattc cccaaattgc gcatccccta 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 cagatgcata tctgtgtaaa tgaaagcatc tgactcaaca ggcatcacat cagataattt
     2641 ttgctgatcg tatgtatggt ctatggcccc tacaaggtca aaatgtggtg ttcgttctgc
     2701 caatttaaca gcttcctgcc ccattcttcc aattgttccc taagtccaac tactaaactg
     2761 ggctaagtcc aactactaaa ctgggccctg aagaagggcc catatagggc cctgaagaag
     2821 ggccctatcg aggatattat ctcgaggctc acctattagc ggctaaggct agagctcgct
     2881 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2941 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3001 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3061 agggggagga ttgggaagac aatagcaggc atgctgggga ggtaaccacg tgcggaccga
     3121 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     3181 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     3241 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     3301 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     3361 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3421 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3481 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3541 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3601 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3661 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     3721 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3781 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3841 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3901 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3961 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     4021 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     4081 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     4141 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     4201 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4261 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     4321 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     4381 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     4441 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4501 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     4561 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     4621 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     4681 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     4741 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     4801 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     4861 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     4921 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     4981 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5041 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     5101 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5161 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5221 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5281 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5341 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     5401 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5461 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5521 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5581 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5641 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5701 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5761 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5821 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
