LOCUS       sequence-319335-        7735 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses NCreIntN in neurons in split intein-mediated split-Cre 
            recombinase applications.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7735)
  AUTHORS   Khoo ATT, Kim PJ, Kim HM, Je HS
  TITLE     Neural circuit analysis using a novel intersectional split 
            intein-mediated split-Cre recombinase system.
  JOURNAL   Mol Brain. 2020 Jul 2;13(1):101. doi: 10.1186/s13041-020-00640-2.
  PUBMED    32616061
REFERENCE   2  (bases 1 to 7735)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol 
            Brain."; date: "2020-07-2"; volume: "13(1):101. doi"; pages: " 
            10.1186/s13041-020-00640-2"
COMMENT     Sequence Label: pCDH-hSyn-NCreIntN-T2A-mTagBFP2
FEATURES             Location/Qualifiers
     source          1..7735
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..181
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    228..353
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    846..1079
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1264..1308
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1575..1691
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1730..2177
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             2202..2222
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2223..2249
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2763..2816
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2823..3533
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             2841..3539
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="ELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVE
                     GGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQ
                     DTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLV
                     GGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDL
                     PSKLGHKLNH"
     misc_feature    3582..4170
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3635..3655)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4053..4064)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4244..4477
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4549..4670
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4586..4605)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4640..4659
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4710..4845
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4878..4894)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4878..4894)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4891..4913)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4902..4918
                     /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)."
     promoter        complement(join(4926..4932,4933..4950,4951..4956))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4971..4992
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5109..5126)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5280..5868)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(6039..6830,6831..6899))
                     /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     6662..6681
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6900..7004)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7072..7090
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7128..7150)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7250..7269
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7463..7485
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7477..7494
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7478..7494
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        7509..7735
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     1501 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatctcga
     1561 cggtatcggt taacttttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga
     1621 atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa
     1681 ttcaaaattt tatcgatatc gatctagact gcagagggcc ctgcgtatga gtgcaagtgg
     1741 gttttaggac caggatgagg cggggtgggg gtgcctacct gacgaccgac cccgacccac
     1801 tggacaagca cccaaccccc attccccaaa ttgcgcatcc cctatcagag agggggaggg
     1861 gaaacaggat gcggcgaggc gcgtgcgcac tgccagcttc agcaccgcgg acagtgcctt
     1921 cgcccccgcc tggcggcgcg cgccaccgcc gcctcagcac tgaaggcgcg ctgacgtcac
     1981 tcgccggtcc cccgcaaact ccccttcccg gccaccttgg tcgcgtccgc gccgccgccg
     2041 gcccagccgg accgcaccac gcgaggcgcg agataggggg gcacgggcgc gaccatctgc
     2101 gctgcggcgc cggcgactca gcgctgcctc agtctgcggt gggcagcgga ggagtcgtgt
     2161 cgtgcctgag agcgcagtcg agaagcggcc gcgccaccat gcccaagaag aagcggaagg
     2221 tgtacccata cgatgttcca gattacgcag gttctggttc caatttactg accgtacacc
     2281 aaaatttgcc tgcattaccg gtcgatgcaa cgagtgatga ggttcgcaag aacctgatgg
     2341 acatgttcag ggatcgccag gcgttttctg agcatacctg gaaaatgctt ctgtccgttt
     2401 gccggtcgtg ggcggcatgg tgcaagttga atgaattcct taaggggtgt ttaagctatg
     2461 aaacggaaat attgacagta gaatatggat cattaccgat tggtaaaatt gtagaaaagc
     2521 gcatcgaatg tactgtttat agtgttgata ataatggaaa tatttataca caacctgtag
     2581 cacaatggca cgatcgcgga gaacaagagg tgtttgagta ttgtttggaa gatggttcat
     2641 tgattcgggc aacaaaagac cataagttta tgactgttga tggtcaaatg ttgccaattg
     2701 atgaaatatt tgaacgtgaa ttggatttga tgcgggttga taatttgccg aattatgcta
     2761 gcgagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc ggccctggat
     2821 ccatggtgtc taagggcgaa gagctgatta aggagaacat gcacatgaag ctgtacatgg
     2881 agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg
     2941 agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg
     3001 acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca
     3061 tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat
     3121 acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca
     3181 tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga
     3241 aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag
     3301 gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca aacgccaaga
     3361 ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg
     3421 actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag cagcacgagg
     3481 tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctt aatcactagg
     3541 gcgcgcccac ctggtgcatg acccgcaagc ccggtgcctg aaatcaacct ctggattaca
     3601 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     3661 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     3721 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     3781 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     3841 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     3901 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     3961 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
     4021 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     4081 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct ccgccttcgc cctcagacga
     4141 gtcggatctc cctttgggcc gcctccccgc ctggtacctt taagaccaat gacttacaag
     4201 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     4261 tcccaacgaa gataagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
     4321 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     4381 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     4441 ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt
     4501 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt
     4561 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4621 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     4681 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4741 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4801 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctagac ttttgcagag
     4861 accaaattcg taatcatgtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4921 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4981 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5041 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     5101 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     5161 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     5221 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5281 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5341 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5401 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5461 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5521 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5581 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5641 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5701 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5761 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5821 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5881 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5941 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6001 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6061 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     6121 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     6181 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6241 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6301 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     6361 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6421 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6481 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6541 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6601 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     6661 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6721 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6781 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6841 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6901 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6961 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7021 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     7081 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     7141 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     7201 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag
     7261 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     7321 aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     7381 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     7441 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgccaag
     7501 ctgacgcgtg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
     7561 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
     7621 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
     7681 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaac
//
