pN1-hCRAFi-CCR2_3KGC_FusionREd_ER2

LOCUS       pN1-hCRAFi-CCR2_3KGC_FusionREd_ER2      6776 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 6776)
  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..6776
                     /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"
     primer_bind     646..692
                     /label=pN1_AgeI_R2_Fw
     misc_feature    673..678
                     /label=Kozak
     misc_feature    679..3438
                     /label=hCRAFi-CCR2_KGC_FusionRed_ER2
     CDS             679..2502
                     /codon_start=1
                     /product="Isoform B"
                     /label=hCCR2B
                     /translation="MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLL
                     PPLYSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAAN
                     EWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVI
                     TWLVAVFASVPGIIFTKCQKEDSVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICY
                     SGILKTLLRCRNGLEEGGNVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIG
                     DGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSK
                     GEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVT
                     TLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVN
                     RIELKGIDFKEDGNILGHKLEYNIDKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTFQ
                     EFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRF
                     CKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL"
     CDS             823..825
                     /codon_start=1
                     /label=Y49A mutation
                     /translation="Y"
     primer_bind     937..957
                     /label=sequencing primer
     CDS             1036..1038
                     /codon_start=1
                     /label=Y120A
                     /translation="Y"
     primer_bind     1300..1317
                     /label=NEW SEQUENCING PRIMER
     CDS             1399..2121
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=cpGFP
                     /translation="NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPV
                     LLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
                     FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
                     GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIEL
                     KGIDFKEDGNILGHKLEYN"
     CDS             2518..2577
                     /codon_start=1
                     /label=KGC
                     /translation="KSRITSEGEYIPLDQIDINV"
     CDS             2578..2637
                     /codon_start=1
                     /label=KGC
                     /translation="KSRITSEGEYIPLDQIDINV"
     CDS             2638..2697
                     /codon_start=1
                     /label=KGC
                     /translation="KSRITSEGEYIPLDQIDINV"
     CDS             2716..3411
                     /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"
     CDS             3418..3438
                     /codon_start=1
                     /label=ER2
                     /translation="FCYENEV"
     polyA_signal    3564..3685
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3687..3726)
                     /label=polyA_long_Rv
     rep_origin      complement(3692..4147)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        4174..4278
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4280..4637
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4488..4623
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4672..5466
                     /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    5698..5745
                     /label=HSV TK poly(A) signal
                     /note="herpesvirus thymidine kinase polyadenylation signal"
     rep_origin      6074..6662
                     /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 gctgagcacc agcagaagca gattcatcag aaacaccaac
      721 gagagcggcg aggaggtgac caccttcttc gactacgact acggcgcccc ctgccacaag
      781 ttcgacgtga agcagatcgg cgcccagctg ctgccccccc tgtacagcct ggtgttcatc
      841 ttcggcttcg tgggcaacat gctggtggtg ctgatcctga tcaactgcaa gaagctgaag
      901 tgcctgaccg acatctacct gctgaacctg gccatcagcg acctgctgtt cctgatcacc
      961 ctgcccctgt gggcccacag cgccgccaac gagtgggtgt tcggcaacgc catgtgcaag
     1021 ctgttcaccg gcctgtacca catcggctac ttcggcggca tcttcttcat catcctgctg
     1081 accatcgaca gatacctggc catcgtgcac gccgtgttcg ccctgaaggc cagaaccgtg
     1141 accttcggcg tggtgaccag cgtgatcacc tggctggtgg ccgtgttcgc cagcgtgccc
     1201 ggcatcatct tcaccaagtg ccagaaggag gacagcgtgt acgtgtgcgg cccctacttc
     1261 cccagaggct ggaacaactt ccacaccatc atgagaaaca tcctgggcct ggtgctgccc
     1321 ctgctgatca tggtgatctg ctacagcggc atcctgaaga ccctgctgag atgcagaaac
     1381 ggcctggagg agggaggaaa cgtctatatc aaggccgaca agcagaagaa cggcatcaag
     1441 gcgaacttca agatccgcca caacatcgag gacggcggcg tgcagctcgc ctaccactac
     1501 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1561 gtgcagtcca aactttcgaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1621 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaaggg cggtaccgga
     1681 gggagcatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1741 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggtga gggcgatgcc
     1801 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1861 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     1921 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacatcca ggagcgcacc
     1981 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     2041 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     2101 gggcacaagc tggagtacaa catcgacaag aagagacaca gagccgtgag agtgatcttc
     2161 accatcatga tcgtgtactt cctgttctgg accccctaca acatcgtgat cctgctgaac
     2221 accttccagg agttcttcgg cctgagcaac tgcgagagca ccagccagct ggaccaggcc
     2281 acccaggtga ccgagaccct gggcatgacc cactgctgca tcaaccccat catctacgcc
     2341 ttcgtgggcg agaagttcag aagatacctg agcgtgttct tcagaaagca catcaccaag
     2401 agattctgca agcagtgccc cgtgttctac agagagaccg tggacggcgt gaccagcacc
     2461 aacaccccca gcaccggcga gcaggaggtg agcgccggcc tgatggacga gctgtacaag
     2521 agtagaatca caagcgaagg cgagtacatc cccctggatc aaatagacat aaatgtaaag
     2581 agtagaatca caagcgaagg cgagtacatc cccctggatc aaatagacat aaatgtaaag
     2641 agtagaatca caagcgaagg cgagtacatc cccctggatc aaatagacat aaatgtaggt
     2701 ggacacgtga ctagtatggt gagcgagctg attaaggaga acatgcccat gaagctgtac
     2761 atggagggca ccgtgaacaa ccaccacttc aagtgcacat ccgagggcga aggcaagccc
     2821 tacgagggca cccagaccat gagaatcaag gtcgtcgagg gcggccctct ccccttcgcc
     2881 ttcgacatcc tggctaccag cttcatgtac ggcagcagaa ccttcatcaa gcaccctccg
     2941 ggcatccccg acttctttaa gcagtccttc cctgagggct tcacatggga gagagtcacc
     3001 acatacgaag acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc
     3061 ctcatctaca acgtcaaggt tagaggggtg aacttcccag ccaacggccc tgtgatgcag
     3121 aagaaaacac tcggctggga ggcctccacc gagacgatgt accctgctga cggaggactg
     3181 gaaggcgcat gtgacatggc tctgaagctc gtgggaggag gacacctgat ctgcaacctt
     3241 gagacaacat acagatccaa gaaacccgct acgaacctca agatgcccgg cgtctacaac
     3301 gtggaccaca gactggaaag aatcaaggag gccgacgatg agacatacgt cgagcagcac
     3361 gaggtggctg tggccagata ctctactggt ggcgctggtg atggaggtaa aggtggattt
     3421 tgttatgaga atgaagtgta aagcggccgc gactctagat cataatcagc cataccacat
     3481 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     3541 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     3601 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3661 tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa
     3721 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     3781 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     3841 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     3901 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     3961 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     4021 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     4081 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     4141 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     4201 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     4261 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     4321 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     4381 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     4441 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     4501 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     4561 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     4621 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     4681 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4741 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4801 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     4861 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4921 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4981 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     5041 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     5101 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     5161 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     5221 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     5281 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     5341 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     5401 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     5461 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     5521 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     5581 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     5641 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     5701 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     5761 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     5821 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     5881 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     5941 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6001 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6061 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6121 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6181 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6241 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6301 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6361 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6421 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6481 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6541 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6601 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6661 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6721 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//