LOCUS       sequence-269554-        6078 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the P4 coiled coil fused to the CIBN blue light-inducible 
            domain in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6078)
  AUTHORS   Lebar T, Lainscek D, Merljak E, Aupic J, Jerala R
  TITLE     A tunable orthogonal coiled-coil interaction toolbox for engineering
            mammalian cells.
  JOURNAL   Nat Chem Biol. 2020 Jan 6. pii: 10.1038/s41589-019-0443-y. doi: 
            10.1038/s41589-019-0443-y.
  PUBMED    31907374
REFERENCE   2  (bases 1 to 6078)
  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: "Nat Chem 
            Biol. 2020 Jan 6. pii: 10.1038/s41589-019-0443-y. doi: 
            10.1038/s41589-019-0443-y."
COMMENT     Sequence Label: pcDNA3-P4:gs:nls:CIBN
FEATURES             Location/Qualifiers
     source          1..6078
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1085..1105
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1106..1612
                     /codon_start=1
                     /product="N-terminal portion of the CIB1 transcription 
                     factor from Arabidopsis thaliana"
                     /label=CIBN
                     /note="binds to CRY2 (cryptochrome 2) that has absorbed 
                     blue light (Kennedy et al., 2010)"
                     /translation="NGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSM
                     ITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKAAKFDTETKDCNE
                     AAKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKEL
                     EKTDYI"
     promoter        complement(1631..1649)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1632..1649)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1669..1686)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1675..1899
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1945..2373
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2032..2051)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2242..2263
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2382..2402)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2387..2716
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2567..2702
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2629..2648
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2783..3577
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2837..2856)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3447..3466
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3751..3872
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3788..3807)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3842..3861
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3921..3937)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3921..3937)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3934..3956)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3945..3961
                     /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(3969..3975,3976..3993,3994..3999))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4014..4035
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4152..4169)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4323..4911)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4403..4422)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5082..5873,5874..5942))
                     /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     5705..5724
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5943..6047)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcgccacca tgagcccgga
      961 agataaaatt gctcagctga aacaaaaaat ccaagcgctg aaacaggaaa accagcagct
     1021 ggaagaggaa aacgccgcac tggaatatgg tggcggctct ggcggcggct ccggaggctc
     1081 tgatccaaaa aagaagagaa aggtaaatgg agctatagga ggtgaccttt tgctcaattt
     1141 tcctgacatg tcggtcctag agcgccaaag ggctcacctc aagtacctca atcccacctt
     1201 tgattctcct ctcgccggct tctttgccga ttcttcaatg attaccggcg gcgagatgga
     1261 cagctatctt tcgactgccg gtttgaatct tccgatgatg tacggtgaga cgacggtgga
     1321 aggtgattca agactctcaa tttcgccgga aacgacgctt gggactggaa atttcaaggc
     1381 agcgaagttt gatacagaga ctaaggattg taatgaggcg gcgaagaaga tgacgatgaa
     1441 cagagatgac ctagtagaag aaggagaaga agagaagtcg aaaataacag agcaaaacaa
     1501 tgggagcaca aaaagcatca agaagatgaa acacaaagcc aagaaagaag agaacaattt
     1561 ctctaatgat tcatctaaag tgacgaagga attggagaaa acggattata tttaatctag
     1621 agggccctat tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc
     1681 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     1741 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     1801 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     1861 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     1921 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1981 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2041 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2101 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2161 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2221 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2281 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2341 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     2401 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     2461 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     2521 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     2581 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     2641 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     2701 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga
     2761 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     2821 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     2881 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     2941 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     3001 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     3061 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     3121 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     3181 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     3241 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     3301 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     3361 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     3421 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     3481 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     3541 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     3601 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     3661 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     3721 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     3781 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     3841 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     3901 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3961 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4021 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4081 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4141 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4201 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4261 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4321 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4381 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4441 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4501 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4561 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4621 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4681 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4741 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4801 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4861 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4921 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4981 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5041 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5101 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5161 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5221 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5281 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5341 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5401 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     5461 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5521 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5581 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5641 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5701 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5761 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5821 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5881 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5941 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6001 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6061 aaagtgccac ctgacgtc
//
