piRFP-PAS-FRB-P2A-FKBP-iRFP-GAF-N1-linker2

LOCUS       piRFP-PAS-FRB-P2A-FKBP-iRFP-GAF-N1-linker2              5923 bp DNA     circular SYN 21-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5923)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..5923
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        363..666
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        667..870
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    893..898
                     /label=MCS
                     /note="multiple cloning site"
     misc_feature    899..904
                     /label=Kozak
     CDS             905..1273
                     /codon_start=1
                     /label=iRFP-PAS
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQR"
     misc_feature    1271..1273
                     /label=Insert
     CDS             1283..1297
                     /codon_start=1
                     /label=Linker
                     /translation="GGGGS"
     CDS             1304..1582
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             1592..1648
                     /codon_start=1
                     /label=P2A
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1649..1969
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1976..1990
                     /codon_start=1
                     /label=Linker
                     /translation="GGGGS"
     CDS             1991..1999
                     /codon_start=1
                     /product="phytochrome-based near-infrared fluorescent 
                     protein (Filonov et al., 2011)"
                     /label=iRFP
                     /translation="CGR"
     CDS             2000..2578
                     /codon_start=1
                     /label=iRFP-GAF
                     /translation="DVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKITGFDRV
                     MIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDINYRPV
                     PVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGLIVCHH
                     RTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     polyA_signal    2707..2828
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2835..3290)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        3317..3421
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3423..3780
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3631..3766
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3815..4609
                     /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"
     polyA_signal    4841..4888
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal"
     rep_origin      5217..5805
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 asaatiiaat iindaatiis nabibtgnca atiibmtinh bgsacihind iiiasaccis
       61 aaccksacii asmaibamhi agncbamhib sbstbbkasi narisbsrbi bbsismaibt
      121 gbcsacibsa atiisbssac iiacuistui bsgianssva nanmunihab tsiaaabtgd
      181 raiiibsrbi sbsrbibsns ssbsnssncs stuicbsbbk asinaristt hacuibsrdi
      241 bsbtgnssnc btgbsrsrii acuibsrbib tgbsrbibsb saiaacuisa bssansircw
      301 nstagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      361 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      421 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      481 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      541 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      601 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      661 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      721 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      781 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      841 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
      901 caccatggcg gaaggatccg tcgccaggca gcctgacctc ttgacctgcg acgatgagcc
      961 gatccatatc cccggtgcca tccaaccgca tggactgctg ctcgccctcg ccgccgacat
     1021 gacgatcgtt gccggcagcg acaaccttcc cgaactcacc ggactggcga tcggcgccct
     1081 gatcggccgc tctgcggccg atgtcttcga ctcggagacg cacaaccgtc tgacgatcgc
     1141 cttggccgag cccggggcgg ccgtcggagc accgatcact gtcggcttca cgatgcgaaa
     1201 ggacgcaggc ttcatcggct cctggcatcg ccatgatcag ctcatcttcc tcgagctcga
     1261 gcctccccag cggtgcggcc gcggcggagg cgggtcctct agaatcctct ggcatgagat
     1321 gtggcatgaa ggcctggaag aggcatctcg tttgtacttt ggggaaagga acgtgaaagg
     1381 catgtttgag gtgctggagc ccttgcatgc tatgatggaa cggggccccc agactctgaa
     1441 ggaaacatcc tttaatcagg cctatggtcg agatttaatg gaggcccaag agtggtgcag
     1501 gaagtacatg aaatcaggga atgtcaagga cctcctccaa gcctgggacc tctattatca
     1561 tgtgttccga cgaatctcaa agggatccgg agctactaac ttcagcctgc tgaagcaggc
     1621 tggagacgtg gaggagaacc ctggacctgg agtgcaggtg gaaaccatct ccccaggaga
     1681 cgggcgcacc ttccccaagc gcggccagac ctgcgtggtg cactacaccg ggatgcttga
     1741 agatggaaag aaatttgatt cctcccggga cagaaacaag ccctttaagt ttatgctagg
     1801 caagcaggag gtgatccgag gctgggaaga aggggttgcc cagatgagtg tgggtcagag
     1861 agccaaactg actatatctc cagattatgc ctatggtgcc actgggcacc caggcatcat
     1921 cccaccacat gccactctcg tcttcgatgt ggagcttcta aaactggaaa ctagtggcgg
     1981 aggcgggtcc tgcggccgcg acgtcgccga gccgcaggcg ttcttccgcc gcaccaacag
     2041 cgccatccgc cgcctgcagg ccgccgaaac cttggaaagc gcctgcgccg ccgcggcgca
     2101 agaggtgcgg aagattaccg gcttcgatcg ggtgatgatc tatcgcttcg cctccgactt
     2161 cagcggcgaa gtgatcgcag aggatcggtg cgccgaggtc gagtcaaaac taggcctgca
     2221 ctatcctgcc tcaaccgtgc cggcgcaggc ccgtcggctc tataccatca acccggtacg
     2281 gatcattccc gatatcaatt atcggccggt gccggtcacc ccagacctca atccggtcac
     2341 cgggcggccg attgatctta gcttcgccat cctgcgcagc gtctcgcccg tccatctgga
     2401 attcatgcgc aacataggca tgcacggcac gatgtcgatc tcgattttgc gcggcgagcg
     2461 actgtgggga ttgatcgttt gccatcaccg aacgccgtac tacgtcgatc tcgatggccg
     2521 ccaagcctgc gagctagtcg cccaggttct ggcctggcag atcggcgtga tggaagagta
     2581 aaagcttgcg gccgactcta gatcataatc agccatacca catttgtaga ggttttactt
     2641 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     2701 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2761 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2821 gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     2881 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2941 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     3001 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     3061 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     3121 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     3181 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     3241 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     3301 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3361 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3421 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3481 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3541 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3601 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3661 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3721 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3781 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3841 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3901 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3961 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     4021 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     4081 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     4141 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     4201 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     4261 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     4321 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     4381 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     4441 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4501 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4561 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4621 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4681 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4741 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     4801 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     4861 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     4921 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     4981 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     5041 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     5101 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5161 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5221 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5281 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5341 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     5401 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5461 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5521 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5581 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5641 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5701 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5761 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5821 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5881 tcccctgatt ctgtggataa ccgtattacc gccatgcatd ann
//