pN1-mCRAFi-CCR2_P2A_FusionREd

LOCUS       pN1-mCRAFi-CCR2_P2A_FusionREd               6890 bp DNA     circular SYN 25-MAR-2025
DEFINITION  PM20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6890)
  AUTHORS   Kiryl Piatkevich
  TITLE     Direct Submission
  JOURNAL   Exported Aug 5, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..6890
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..53
                     /label=CMV_long
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    591..666
                     /label=MCS
                     /note="multiple cloning site of fluorescent protein 
                     plasmids"
     misc_feature    673..678
                     /label=Kozak
     CDS             679..2541
                     /codon_start=1
                     /label=mCCR2
                     /translation="MEDNNMLPQFIHGILSTSHSLFTRSIQELDEGATTPYDYDDGEPC
                     HKTSVKQIGAWILPPLYSLVFIFGFVGNMLVIIILIGCKKLKSMTDIYLLNLAISDLLF
                     LLTLPFWAHYAANEWVFGNIMCKVFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALK
                     ARTVTFGVITSVVTWVVAVFASLPGIIFTKSKQDDHHYTCGPYFTQLWKNFQTIMRNIL
                     SLILPLLVMVICYSGILHTLFRCRNGLEEGGNVYIKADKQKNGIKANFKIRHNIEDGGV
                     QLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDE
                     LYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT
                     TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKT
                     RAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNIDKKRHRAVRLIFAIMIVYFLFW
                     TPYNIVLFLTTFQESLGMSNCVIDKHLDQAMQVTETLGMTHCCINPVIYAFVGEKFRRY
                     LSIFFRKHIAKRLCKQCPVFYRETADRVSSTFTPSTGEQEVSVGL"
     CDS             844..927
                     /codon_start=1
                     /label=TM1
                     /translation="WILPPLYSLVFIFGFVGNMLVIIILIGC"
     primer_bind     865..889
                     /label=Y49A_new_Fw
     CDS             958..1020
                     /codon_start=1
                     /label=TM2
                     /translation="LLNLAISDLLFLLTLPFWAHY"
     primer_bind     complement(1053..1098)
                     /label=Y120A_Rv_new
     CDS             1060..1125
                     /codon_start=1
                     /label=TM3
                     /translation="VFTGLYHIGYFGGIFFIILLTI"
     primer_bind     1078..1098
                     /label=Y120A_new_Fw
     CDS             1177..1251
                     /codon_start=1
                     /label=TM4
                     /translation="VTFGVITSVVTWVVAVFASLPGIIF"
     CDS             1336..1395
                     /codon_start=1
                     /label=TM5
                     /translation="NILSLILPLLVMVICYSGIL"
     primer_bind     complement(1396..1437)
                     /label=mR2_NLEG_Rv
     primer_bind     1420..1464
                     /label=NLEG_GFP_Fw
     misc_feature    1420..1437
                     /label=mut10_P2A_FUsionREd
     primer_bind     1423..1464
                     /label=LEGG_GFP_Fw
     misc_feature    1438..2166
                     /label=ICL3
     CDS             1438..2166
                     /codon_start=1
                     /label=linker+cpGFP
                     /translation="NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPV
                     LLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
                     FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
                     GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL
                     KGIDFKEDGNILGHKLEYNID"
     CDS             1438..2160
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=cpGFP
                     /translation="NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPV
                     LLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
                     FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
                     GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL
                     KGIDFKEDGNILGHKLEYN"
     CDS             2191..2265
                     /codon_start=1
                     /label=TM6
                     /translation="LIFAIMIVYFLFWTPYNIVLFLTTF"
     CDS             2317..2388
                     /codon_start=1
                     /label=TM7
                     /translation="AMQVTETLGMTHCCINPVIYAFVG"
     primer_bind     complement(2521..2541)
                     /label=mR2_Rv
     primer_bind     2523..2573
                     /label=mR2_KGC_Fw
     CDS             2560..2616
                     /codon_start=1
                     /label=KGC
                     /translation="SRITSEGEYIPLDQIDINV"
     primer_bind     complement(2589..2616)
                     /label=KGC_GG_Rv
     CDS             2617..2676
                     /codon_start=1
                     /label=KGC
                     /translation="KSRITSEGEYIPLDQIDINV"
     primer_bind     2617..2633
                     /label=mR2_KGC_Fw
     primer_bind     complement(2649..2676)
                     /label=KGC_GG_Rv
     CDS             2677..2736
                     /codon_start=1
                     /label=KGC
                     /translation="KSRITSEGEYIPLDQIDINV"
     primer_bind     2677..2693
                     /label=mR2_KGC_Fw
     primer_bind     complement(2709..2742)
                     /label=KGC_GG_Rv
     primer_bind     complement(2737..2763)
                     /label=GGER2_NotI_Rv
     CDS             2743..2763
                     /codon_start=1
                     /label=ER2
                     /translation="FCYENEV"
     CDS             2788..2844
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     2844..2871
                     /label=GGFusionRed_Fw
     primer_bind     complement(2844..2859)
                     /label=KGC_GG_Rv
     CDS             2857..3552
                     /codon_start=1
                     /product="monomeric red fluorescent protein optimized for 
                     use as a fusion tag (Shemiakina et al., 2012)"
                     /label=FusionRed
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMPMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFMYGSRTFIKHPPGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKVRGVNFPANGPVMQKKTLGWEASTETMYPADGGLEGACDMAL
                     KLVGGGHLICNLETTYRSKKPATNLKMPGVYNVDHRLERIKEADDETYVEQHEVAVARY
                     STGGAGDGGK"
     primer_bind     complement(2934..2953)
                     /label=p2A_Fusionred_seq_Rv
     primer_bind     complement(3529..3560)
                     /label=FusionRed_GGER2_Rv
     primer_bind     complement(3532..3583)
                     /label=pN1_FusionREd_NotI_Rv
     primer_bind     complement(3553..3583)
                     /label=GGER2_NotI_Rv
     primer_bind     complement(3560..3590)
                     /label=pN1_Not_Rv
     polyA_signal    3678..3799
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3782..3812)
                     /label=N1-CMV-Rv
     primer_bind     complement(3797..3817)
                     /label=N1-CMV-Rv
     primer_bind     complement(3801..3840)
                     /label=polyA_long_Rv
     rep_origin      complement(3806..4261)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        4288..4392
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4394..4751
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4602..4737
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4786..5580
                     /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)"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     misc_feature    5812..5859
                     /label=HSV TK poly(A) signal
                     /note="herpesvirus thymidine kinase polyadenylation signal"
     rep_origin      6188..6776
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccaccgg tcgccaccat ggaggacaac aacatgctgc ctcagttcat ccacggcatc
      721 ctgagcacca gccacagcct gttcaccagg agcatccagg agctggacga gggcgccacc
      781 accccttacg actacgacga cggcgagcct tgccacaaga ccagcgtgaa gcagatcggc
      841 gcctggatcc tgcctcctct gtacagcctg gtgttcatct tcggcttcgt gggcaacatg
      901 ctggtgatca tcatcctgat cggctgcaag aagctgaaga gcatgaccga catctacctg
      961 ctgaacctgg ccatcagcga cctgctgttc ctgctgaccc tgcctttctg ggcccactac
     1021 gccgccaacg agtgggtgtt cggcaacatc atgtgcaagg tgttcaccgg cctgtaccac
     1081 atcggctact tcggcggcat cttcttcatc atcctgctga ccatcgacag gtacctggcc
     1141 atcgtgcacg ccgtgttcgc cctgaaggcc aggaccgtga ccttcggcgt gatcaccagc
     1201 gtggtgacct gggtggtggc cgtgttcgcc agcctgcctg gcatcatctt caccaagagc
     1261 aagcaggacg accaccacta cacctgcggc ccttacttca cccagctgtg gaagaacttc
     1321 cagaccatca tgaggaacat cctgagcctg atcctgcctc tgctggtgat ggtgatctgc
     1381 tacagcggca tcctgcacac cctgttcagg tgcaggaacg gcctggagga gggaggaaac
     1441 gtctatatca aggccgacaa gcagaagaac ggcatcaagg cgaacttcaa gatccgccac
     1501 aacatcgagg acggcggcgt gcagctcgcc taccactacc agcagaacac ccccatcggc
     1561 gacggccccg tgctgctgcc cgacaaccac tacctgagcg tgcagtccaa actttcgaaa
     1621 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1681 actctcggca tggacgagct gtacaagggc ggtaccggag ggagcatggt gagcaagggc
     1741 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1801 cacaagttca gcgtgtccgg cgagggtgag ggcgatgcca cctacggcaa gctgaccctg
     1861 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1921 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1981 aagtccgcca tgcccgaagg ctacatccag gagcgcacca tcttcttcaa ggacgacggc
     2041 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     2101 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     2161 atcgacaaga agaggcacag ggccgtgagg ctgatcttcg ccatcatgat cgtgtacttc
     2221 ctgttctgga ccccttacaa catcgtgctg ttcctgacca ccttccagga gagcctgggc
     2281 atgagcaact gcgtgatcga caagcacctg gaccaggcca tgcaggtgac cgagaccctg
     2341 ggcatgaccc actgctgcat caaccctgtg atctacgcct tcgtgggcga gaagttcagg
     2401 aggtacctga gcatcttctt caggaagcac atcgccaaga ggctgtgcaa gcagtgccct
     2461 gtgttctaca gggagaccgc cgacagggtg agcagcacct tcacccctag caccggcgag
     2521 caggaggtga gcgtgggcct gatggacgag ctgtacaaga gtagaatcac aagcgaaggc
     2581 gagtacatcc ccctggatca aatagacata aatgtaaaga gtagaatcac aagcgaaggc
     2641 gagtacatcc ccctggatca aatagacata aatgtaaaga gtagaatcac aagcgaaggc
     2701 gagtacatcc ccctggatca aatagacata aatgtaggtg gattttgtta tgagaatgaa
     2761 gtgcgaccgg ttcacgtggg aagtggggct acaaacttct cactgctgaa acaggccggg
     2821 gatgtggagg aaaacccagg accacacgtg actagtatgg tgagcgagct gattaaggag
     2881 aacatgccca tgaagctgta catggagggc accgtgaaca accaccactt caagtgcaca
     2941 tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa ggtcgtcgag
     3001 ggcggccctc tccccttcgc cttcgacatc ctggctacca gcttcatgta cggcagcaga
     3061 accttcatca agcaccctcc gggcatcccc gacttcttta agcagtcctt ccctgagggc
     3121 ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc tacccaggac
     3181 accagcctcc aggacggctg cctcatctac aacgtcaagg ttagaggggt gaacttccca
     3241 gccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggcctccac cgagacgatg
     3301 taccctgctg acggaggact ggaaggcgca tgtgacatgg ctctgaagct cgtgggagga
     3361 ggacacctga tctgcaacct tgagacaaca tacagatcca agaaacccgc tacgaacctc
     3421 aagatgcccg gcgtctacaa cgtggaccac agactggaaa gaatcaagga ggccgacgat
     3481 gagacatacg tcgagcagca cgaggtggct gtggccagat actctactgg tggcgctggt
     3541 gatggaggta aataaagcgg ccgcgactct agatcataat cagccatacc acatttgtag
     3601 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     3661 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3721 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3781 aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     3841 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     3901 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     3961 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     4021 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     4081 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     4141 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     4201 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     4261 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4321 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4381 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     4441 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4501 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4561 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     4621 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4681 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4741 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4801 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4861 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4921 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     4981 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     5041 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     5101 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     5161 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     5221 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     5281 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     5341 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     5401 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     5461 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     5521 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     5581 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     5641 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5701 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     5761 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     5821 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     5881 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     5941 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     6001 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
     6061 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6121 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6181 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6241 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6301 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6361 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6421 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6481 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6541 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6601 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6661 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6721 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6781 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6841 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//