LOCUS       sequence-321632-        7988 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses IntCCCre, tagged with FLAG, in mammalian cells in split 
            intern-mediated split-Cre recombinase applications.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7988)
  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 7988)
  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: "Mol 
            Brain."; date: "2020-07-2"; volume: "13(1):101. doi"; pages: " 
            10.1186/s13041-020-00640-2"
COMMENT     Sequence Label: FLAG-IntCCCre
FEATURES             Location/Qualifiers
     source          1..7988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2283..2306
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3333..3356
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    join(3374..3948,3949..3951,3952..3960)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3541..3558)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3768..3787
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3961..3963,3964..3966,3967..4680)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3961..3963,3964..3966,3967..4677)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4267..4286)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4614..4635
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    4803..4924
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4840..4859)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4894..4913
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4931..5386)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5068..5089)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5280..5299
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5413..5517
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5519..5876
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5519..5710
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5542..5562)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5727..5862
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5911..6705
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             5911..6705
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5965..5984)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6575..6594
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6893..6912)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6937..6984
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      7313..7901
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7802..7821
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtggataacc gtattaccgc catgcattag ttattaatga gctcggagga cacttgctgg
       61 gagccttggt gtttgtaact ccgacttgga atcaaggcca cattagaatg cagcaaagca
      121 tgaacattca ggaaagagag atcttacttc aagttccggg aatatccagg tgtagctttt
      181 cttttcaaaa ctccctggtc cgttagtcag atcccctgag ccataacttc caccgctccc
      241 caataactct cctgtagcat cccctaaaat ggagaggtga gagccagaat ccgagttaca
      301 tttcactgaa tgaggatatg ccaactcttc atagaagggc tgtcccaata aaatctgcct
      361 ctactgtgct taaccttcaa gatttttctg cggatccagg tatttaaacg caacacccag
      421 agtaaatgaa aaaaaacaaa aacaaaacaa aaaaacaacc ccccccccca aaaaaaaaga
      481 caatgctcct tgcttgctct gctctcagcc agttgacaga acaaagaaaa agaaaaactc
      541 caggcagttt atctctagaa tcccagaatg attggtgcca tgttttaggc caccaaagaa
      601 ctcccacaat gccttgcttt tctttttaga ttcgttccgg gtctggaagg ttactttcaa
      661 tatcttgcaa atgcccagga tctccttaga accctctccc cctccccttt cccagatgta
      721 ccccattcca gccactcaac cctgacactg tttgcaaact caagggcaat tgcctttaca
      781 taagcaattc aagtaggatg cattcattgc aaaggggttg aagagggtaa gaacttaatg
      841 aagagttaga agaggaactg gaaagagaaa ggaggccaac cattcctcct cctcctctgt
      901 cacttttctt gggctagaag gacggaggat catccatctg aaagattcct ccttcagatg
      961 gagaaaataa agactcctta tttaagaata gataaggcta ttccacaata gactttgctt
     1021 gactcaaaac acagagtccc cagaaaggga aacagcacat tgaatagagt tttattgcag
     1081 cttctctttg agcttcttct gaccaaggcc ccagagattg agcactaggg gctcaaggaa
     1141 ggctgtgatt gaattatggt ctgtgtcaga gatgaaattc ccacctctgg tgcggggttg
     1201 tttaatctta gcattcccta gatgagctag ctgaggtcat tcattactcc aggagcatct
     1261 gaaaaccaac ggagccaagg tctgctggca aaaactagct ttagacacaa aattctaggg
     1321 tttagagttg aagactctta gtgaagtctc tggggtagga gcaggtgacc gttaggttta
     1381 catttggagt gttcaagtgt agggtatgga agctgacttg caaaggattc tagaagatgc
     1441 tctagtcagg aaggttgagt caaggctgtg tttccagatg aggtcgtttt tatatgaaat
     1501 aaatgtttgc ctagagaggt gatggagatt agaagccagc caccctttgg tggcaataag
     1561 caaagcctct gctaagaggt agaagctaag agatgggcct ctccagagag aacaaagtca
     1621 aactgcacca ccggagacca ttgtcaaggg aaggaccctt cagtgcacct cagtatctgg
     1681 gattgtgagt ggttcacctc tgtctacctc tctgcgctaa agcagtcttc aggctaggac
     1741 atagactcag tgatctttct ttacctttcc agctcgcctc ccgcaacccc ccccccccca
     1801 catgtcctct gtcttctgct gccacaaagg gttaatctct cctgcgggta aaaacaggtc
     1861 cgcggagtct ctaactggcg acagatgggc cactttcttc tggccacaaa ggggccggaa
     1921 tggagcgctc cgcggcatac aaataggcag gtcacgtggt tcccggctct tggctggacc
     1981 gggaagacca tatggcgcat gccggggagg aaggagcagg ggcggaggta ctgtgggggt
     2041 gaggggagtg gtggtggaag ggggcgggag gaaagttctg gggaggggtg aatgagggcg
     2101 ggagtcgttc agtctggacg cgtgcgcgat tgcggggcgc agggacctgc tcgttgctca
     2161 gctcacggcc ccgcccgcgc tcagcatcag caactcggct atataaccct agcgcccgcg
     2221 ccaccgggac acgaggaatt cctcgagctc aagcttcgaa ttctgcagtc gacaccgcca
     2281 tggattataa agacgatgac gataaaatgg ttaaagttat cggtcgtcgt tccctcggag
     2341 tgcaaagaat atttgatatt ggtcttcgcc aagaccataa ttttctgcta gccaatgggg
     2401 cgatcgcggc caattgcttc aataaggagg acggatccaa ccggaaatgg tttcccgcag
     2461 aacctgaaga tgttcgcgat tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa
     2521 ctatccagca acatttgggc cagctaaaca tgcttcatcg tcggtccggg ctgccacgac
     2581 caagtgacag caatgctgtt tcactggtta tgcggcggat acgaaaagaa aacgttgatg
     2641 ccggtgaacg tgcaaaacag gctctagcgt tcgaacgcac tgatttcgac caggttcgtt
     2701 cactcatgga aaatagcgat cgctgccagg atatacgtaa tctggcattt ctggggattg
     2761 cttataacac cctgttacgt atagccgaaa ttgccaggat cagggttaaa gatatctcac
     2821 gtactgacgg tgggagaatg ttaatccata ttggcagaac gaaaacgctg gttagcaccg
     2881 caggtgtaga gaaggcactt agcctggggg taactaaact ggtcgagcga tggatttccg
     2941 tctctggtgt agctgatgat ccgaataact acctgttttg ccgggtcaga aaaaatggtg
     3001 ttgccgcgcc atctgccacc agccagctat caactcgcgc cctggaaggg atttttgaag
     3061 caactcatcg attgatttac ggcgctaagg atgactctgg tcagagatac ctggcctggt
     3121 ctggacacag tgcccgtgtc ggagccgcgc gagatatggc ccgcgctgga gtttcaatac
     3181 cggagatcat gcaagctggt ggctggacca atgtaaatat tgtcatgaac tatatccgta
     3241 acctggatag tgaaacaggg gcaatggtgc gcctgctgga agatggcgat gaattctgca
     3301 gatatccatc acactggcgg ccgctcgaga tggactacaa ggacgacgat gataagggct
     3361 gacccgggat ccgcccctct ccctcccccc cccctaacgt tactggccga agccgcttgg
     3421 aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca
     3481 atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc
     3541 ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag
     3601 cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg
     3661 gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac
     3721 aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa
     3781 gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc
     3841 tggggcctcg gtacacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc
     3901 ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat ggccacaacc
     3961 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     4021 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     4081 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     4141 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
     4201 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     4261 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     4321 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     4381 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     4441 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     4501 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     4561 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     4621 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     4681 agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     4741 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     4801 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4861 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4921 cttaaggcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     4981 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     5041 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     5101 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     5161 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     5221 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     5281 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     5341 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt
     5401 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5461 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct
     5521 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     5581 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     5641 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     5701 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     5761 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     5821 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc
     5881 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     5941 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     6001 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     6061 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
     6121 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     6181 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     6241 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     6301 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     6361 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     6421 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     6481 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     6541 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     6601 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     6661 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     6721 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     6781 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     6841 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac
     6901 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa
     6961 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     7021 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     7081 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     7141 ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat
     7201 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     7261 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     7321 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     7381 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     7441 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     7501 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     7561 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     7621 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     7681 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     7741 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7801 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7861 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7921 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     7981 ctgattct
//
