LOCUS       sequence-177056-        7612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express I-PhoCl-HCVp with N-term EBFP2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7612)
  AUTHORS   Zhang W, Lohman AW, Zhuravlova Y, Lu X, Wiens MD, Hoi H, Yaganoglu 
            S, Mohr MA, Kitova EN, Klassen JS, Pantazis P, Thompson RJ, Campbell
            RE
  TITLE     Optogenetic control with a photocleavable protein, PhoCl.
  JOURNAL   Nat Methods. 2017 Mar 13. doi: 10.1038/nmeth.4222.
  PUBMED    28288123
REFERENCE   2  (bases 1 to 7612)
  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 
            Methods. 2017 Mar 13. doi: 10.1038/nmeth.4222."
COMMENT     Sequence Label: pcDNA-I-PhoCl-HCVp
FEATURES             Location/Qualifiers
     source          1..7612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        164..543
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        544..747
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     698..718
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     792..811
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        792..810
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      835..844
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(853..855,856..858,859..1569)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(898..919)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             1738..2463
                     /codon_start=1
                     /product="photocleavable protein that separates into two 
                     fragments upon exposure to violet light (Zhang et al., 
                     2017)"
                     /label=PhoCl
                     /translation="VIPDYFKQSFPEGYSWERSMTYEDGGICIATNDITMEGDSFINKI
                     HFKGTNFPPNGPVMQKRTVGWEASTEKMYERDGVLKGDVKMKLLLKGGGHYRCDYRTTY
                     KVKQKPVKLPDYHFVDHRIEILSHDKDYNKVKLYEHAVARNSTDSMDELYKGGSGGMVS
                     KGEETITSVIKPDMKNKLRMEGNVNGHAFVIEGEGSGKPFEGIQTIDLEVKEGAPLPFA
                     YDILTTAFHYGNRVFTKYPR"
     primer_bind     complement(3132..3149)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3138..3362
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3408..3836
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3495..3514)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3705..3726
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3845..3865)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3850..4179
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4030..4165
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4092..4111
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4246..5040
                     /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(4300..4319)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4910..4929
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5214..5335
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5251..5270)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5305..5324
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5384..5400)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5384..5400)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5397..5419)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5408..5424
                     /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(5432..5438,5439..5456,5457..5462))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5477..5498
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5615..5632)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5786..6374)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5866..5885)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6545..7336,7337..7405))
                     /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     7168..7187
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7406..7510)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7585..7604)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
       61 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
      121 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
      181 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      241 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      301 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      361 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      421 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      481 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      541 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      601 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      661 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      721 cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg
      781 cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcg tgccgccacc
      841 atggcctcga gaatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      901 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgaggggcga gggcgagggc
      961 gatgccacca acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1021 ccctggccca ccctcgtgac caccctgagc cacggcgtgc agtgcttcgc ccgctacccc
     1081 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1141 cgcaccatct tcttcaagga cgacggcacc tacaagaccc gcgccgaggt gaagttcgag
     1201 ggcgacaccc tggtgaaccg catcgagctg aagggcgtcg acttcaagga ggacggcaac
     1261 atcctggggc acaagctgga gtacaacttc aacagccaca acatctatat catggccgtc
     1321 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacgtgga ggacggcagc
     1381 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1441 cccgacagcc actacctgag cacccagtcc gtgctgagca aagaccccaa cgagaagcgc
     1501 gatcacatgg tcctgctgga gttccgcacc gccgccggga tcactctcgg catggacgag
     1561 ctgtacaagg aattcagcgg gtcaggcgga tctggaagtg gtggtggaga acttgatgaa
     1621 ttggtatact tactagatgg gccaggttat gaccctatac atagtggcgg tagtggggga
     1681 tctggtggct caggagggag cggatccatg gtgtccaagg gcgaagagac aaccatagtg
     1741 atccctgact acttcaagca gagcttcccc gagggctaca gctgggagcg cagcatgacc
     1801 tacgaggacg gcggcatctg catcgccacc aacgacatca caatggaggg ggacagcttc
     1861 atcaacaaga tccacttcaa gggcacgaac ttccccccca acggccccgt gatgcagaag
     1921 aggaccgtgg gctgggaggc cagcaccgag aagatgtacg agcgcgacgg cgtgctgaag
     1981 ggcgacgtga agatgaagct gctgctgaag ggcggcggcc actatcgctg cgactaccgc
     2041 accacctaca aggtcaagca gaagcccgta aagctgcccg actaccactt cgtggaccac
     2101 cgcatcgaga tcctgagcca cgacaaggac tacaacaagg tgaagctgta cgagcacgcc
     2161 gtggcccgca actccaccga cagcatggac gagctgtaca agggtggcag cggtggcatg
     2221 gtgagcaagg gcgaggagac cattacaagc gtgatcaagc ctgacatgaa gaacaagctg
     2281 cgcatggagg gcaacgtgaa cggccacgcc ttcgtgatcg agggcgaggg cagcggcaag
     2341 cccttcgagg gcatccagac gattgatttg gaggtgaagg agggcgcccc gctgcccttc
     2401 gcctacgaca tcctgaccac cgccttccac tacggcaacc gcgtgttcac caagtaccca
     2461 cggggaggta gcaccgacgg catggacgag ctgtacaagt ctagaggcag tggaacaggg
     2521 ggttctgttg ttattgttgg tagaattatt ttatctggta gtggtagtat cacggcctac
     2581 tcccaacaga cgcggggcct acttggttgc atcatcacta gcctcacagg ccgggacaag
     2641 aaccaggtcg aaggggaggt tcaagtggtt tctaccgcaa cacaatcttt cctggcgacc
     2701 tgcgtcaacg gcgtgtgctg gactgtctac catggcgctg gctcgaagac cctagccggt
     2761 ccaaaaggtc caatcaccca aatgtacacc aatgtagacc aggacctcgt cggctggcag
     2821 gcgcctccag gggcgcgctc cttgacacca tgcacctgtg gcagctcgga cctttacttg
     2881 gtcacgagac atgctgatgt cattccggtg cgccggcgag gcgacagcag gggaagtcta
     2941 ctctccccca ggcccgtctc ctacctgaaa ggctcctcgg gtggtccatt gctttgccct
     3001 tcggggcacg ctgtgggcat cttccgggct gctgtgtgca cccggggggt cgcgaaggcg
     3061 gtggacttcg tgcccgttga gtctatggaa actaccatgc ggtcttaaaa gcttggtaaa
     3121 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     3181 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3241 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3301 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3361 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     3421 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3481 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3541 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3601 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     3661 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3721 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3781 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     3841 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3901 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3961 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     4021 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4081 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     4141 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     4201 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4261 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4321 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4381 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4441 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4501 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4561 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4621 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4681 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4741 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     4801 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4861 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4921 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4981 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     5041 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     5101 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5161 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     5221 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     5281 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     5341 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     5401 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5461 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5521 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5581 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5641 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5701 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5761 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5821 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5881 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5941 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tataggtatc
     6001 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6061 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6121 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6181 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6241 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6301 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6361 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6421 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6481 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6541 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6601 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6661 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6721 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6781 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6841 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6901 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6961 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7021 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7081 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7141 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7201 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7261 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7321 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7381 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7441 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7501 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     7561 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt ta
//
