LOCUS       sequence-494688-        7647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The set of SynBar constructs used in the optimized connectome-seq 
            experiments..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7647)
  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 7647)
  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-DIO-PostSynBar-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..7647
                     /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        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(660..693)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(744..777)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(799..1509)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1077..1096)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1346..1364
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      1506..1515
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1534..1590)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(1609..1674)
                     /codon_start=1
                     /product="N-terminal RNA-binding domain from the lambda 
                     bacteriophage antiterminator protein N (Baron-Benhamou et 
                     al., 2004)"
                     /label=lambda N peptide
                     /translation="MDAQTRRRERRAEKQAQWKAAN"
     CDS             complement(2317..2364)
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     regulatory      3694..3703
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(4198..4224)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(4231..4257)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(4258..4302)
                     /codon_start=1
                     /product="leader sequence from mouse preprotrypsin"
                     /label=preprotrypsin leader
                     /translation="MSALLILALVGAAVA"
     protein_bind    4316..4349
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    4400..4433
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4503..4523)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4910..5050
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4910..5039
                     /label=AAV2 ITR
     rep_origin      5125..5580
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5212..5231)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5422..5443
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5597..5616)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5716..5738
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5776..5794)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5862..5966
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5967..6035,6036..6827)
                     /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(6185..6204)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6998..7586
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7487..7506
                     /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 gcgtgtgtct agactgcaga gggccctgcg tatgagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgagaag gtaccggatc ctctagagtc gactccggaa
      661 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
      721 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
      781 gcgcctaccg gtactagttt acttgtacag ctcgtccatg ccgccggtgg agtggcggcc
      841 ctcggcgcgt tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg tgatgtccaa
      901 cttgatgttg acgttgtagg cgccgggcag ctgcacgggc ttcttggcct tgtaggtggt
      961 cttgacctca gcgtcgtagt ggccgccgtc cttcagcttc agcctctgct tgatctcgcc
     1021 cttcagggcg ccgtcctcgg ggtacatccg ctcggaggag gcctcccagc ccatggtctt
     1081 cttctgcatt acggggccgt cggaggggaa gttggtgccg cgcagcttca ccttgtagat
     1141 gaactcgccg tcctgcaggg aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa
     1201 gttcatcacg cgctcccact tgaatccctc ggggaaggac agcttcaagt agtcggggat
     1261 gtcggcgggg tgcttcacgt aggccttgga gccgtacatg aactgagggg acaggatgtc
     1321 ccaggcgaag ggcagggggc cacccttggt caccttcagc ttggcggtct gggtgccctc
     1381 gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca cggagccctc
     1441 catgtgcacc ttgaagcgca tgaactcctt gatgatggcc atgttatcct cctcgccctt
     1501 gctcaccatg gtggcgaccg gtggatcccg agcaggtcca gggttctcct ccacgtctcc
     1561 agcctgcttc agcaggctga agttagtagc tccgcttccc atgaattcgt ttgcagcttt
     1621 ccattgagct tgtttctcag cgcgacgctc acgtcgtcgt gtttgtgcgt ccattaccct
     1681 ggttgttgaa tgtgaatggg ggtgagggtg ggggtggggc agtgtattat tctgtcctcc
     1741 agtaaatgtg ttgaatgtat gtaagggctg aatccctggt atagtgttgg gaatcattgt
     1801 gatggtgtta ggtgtcatta aaggaacatc atccggtgac ctcctcatag caagagtata
     1861 atctgggggg caggcggtcc gaagaaccac ctcatgtgga tggatggact cacactcgtg
     1921 atccaagtca gtgtgcttca tttggagaga cataatttcc tcttctggag catgggttag
     1981 gtcgttggtc gtggtgcgct gagggctgca cctccggtgg acatcatgtc tcctcttgtc
     2041 tttcttgtag tatagggctg caaaagccaa gatgttcaga aacagcagag aggctccaac
     2101 tgcgattgtg acacttagct ctgtggagta gtccctttga tctaccgaga agggacttgg
     2161 ttgttgtttg ggatcatcct gcttggcagt gggaaaggct gatgtgactg gagtggaatt
     2221 tttccttgta ggtctgagag tgatgtctgt tgatggcact ttagttgttg tcgaggtata
     2281 acgcgtacct ccagaaccac ctccagaacc tccacctgta attcctgcag cattcacgta
     2341 ttcgtgcaaa accatgtgat ctcttcctcc tcctgaacct ccacctgaac ctccacgcgt
     2401 ctgagaaatg tcattgagat tatgcagatg aggtaccagc tccaaccaga gatttacctt
     2461 attggctctg taatgttctt taactctcgg ttttaatcca atatggagat aaagttggtc
     2521 tttctgggaa tatctggtcc atgctacttc ttcaaagcgg ttgggtttgg tatgaatgaa
     2581 ttttgtgtct tgaggaactg gttgatttgg gtcaccggtt ttagcaaaat tcgtccagta
     2641 tgtcattact acagcactca acatcacatc attcttggag aaattgcaag gaaataactc
     2701 tgtagggcca atcatgggga ttcccaatac atagggaacc tcatccccat gagctgcatc
     2761 agcccaagct ggaacttggt cagtttggca atgatgataa aaggcataga agtatgtagg
     2821 tgagccaaag ttcgagtgaa ggtctgctgt agccacagcg ggtgctaccc actgatggtc
     2881 tgtaaacaaa gccaacaatg tcttccttct agtttcaggg ttatggcgat cagcccagtc
     2941 agtatacatg aatttaatgg tttctctcaa aacatctttg ccttcaggat atccatataa
     3001 attatcaaca aaattagaga cagcaaagtc aaaatcactg gctgatacac catcatcact
     3061 atctactatg ttttccacaa acttcaaccc ttccccttgg ttaactccta acatgatatc
     3121 ataattgagg aactctcctt gttccatcag tatctgaggg tcatctggta ttacatcacc
     3181 atcgatcaca ggtccaaagg ctatgtggta tcgggctggt tgaacatctt gatcaacaag
     3241 ttctttgtaa ggcttcttct gcaggcattc tactaactct actgtatctg acacattgca
     3301 gccaactttt gtggctagaa ttctagcgta ttttgcaggc tggaaactaa cagcccagct
     3361 ggaaagggct gttccactct gagctattgc tcgttgaaaa agtccttttt cagaataatg
     3421 ggataaagtc agcagattga cacatgaacc cccagcgcct gatccaaaca cggtgattcg
     3481 caaggggtca ccaccaaaga acccaatgtt ctcgctggtc catctgaggg cctggatgag
     3541 gtcaaggagc ccgtagtttc ctttggcagc ctgatcccct gtgctcaaga agccaagtac
     3601 cccaagccga tagttgactg tgatgacgat cacattaccg tagcttgcca acacactccc
     3661 atcatataga tttccagtac cttccatgta agagccacca tggatgtaca ccatcactgg
     3721 tttgggaccc ccactgtccc gaatatctcc ttttctacat attttcttgc cgggttttct
     3781 ggcacattcc ttggatattc tttttacatc ttcagttggg acatagatgt ttaagtatag
     3841 acagtcttca ctctggtctt gcacatatga ggaaaccaca tccaagttat tagtgaacca
     3901 cacaggaagc ataacttcag gcaatctgcc atcaatgata ttctggggac atacaggagc
     3961 aaactgagtg gcattccgga tatcagacca gggagatggt ggttctggag gctgaaatcg
     4021 atgttctcct gttggtggag cagcatatgg aaccccaaga aactgaatga caggccccaa
     4081 aatttcatta ttgagttctt tcttaattcc ccttattttg ccaaagttag tagtaaccaa
     4141 tgggtctaca tcatccaact tttgcagatc tatcgatgaa ttcgcggccg caagcttggc
     4201 atagtcaggc acgtcataag gatagctagc agcgtaatct ggaacatcgt atgggtaagc
     4261 aactgcagct ccaacaagag ctaggatcag aagtgcagac atggtggcgg ctagcataac
     4321 ttcgtataaa gtatcctata cgaagttatt tgccttaacc cagaaattat cactgttatt
     4381 ctttagaatg gtgcaaagaa taacttcgta taatgtatgc tatacgaagt tatgaattcc
     4441 gctcgagata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     4501 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     4561 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttagt tcttgccacg
     4621 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     4681 gacaattccg tggtgtttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatc
     4741 tagctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     4801 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg
     4861 ggaggttttt taaagcggcc gcaggaacac gtgcggaccg agcggccgca ggaaccccta
     4921 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     4981 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     5041 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     5101 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5161 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     5221 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5281 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5341 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5401 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     5461 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     5521 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     5581 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     5641 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     5701 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     5761 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     5821 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5881 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5941 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     6001 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     6061 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     6121 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     6181 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     6241 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6301 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6361 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6421 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6481 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6541 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6601 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6661 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6721 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6781 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6841 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6901 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6961 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     7021 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     7081 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     7141 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     7201 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     7261 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     7321 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7381 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7441 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7501 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7561 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7621 ccttttgctg gccttttgct cacatgt
//
