pN1-hCRAFi-CCR2null_P2A_FusionRed

LOCUS       pN1-hCRAFi-CCR2null_P2A_FusionRed              6851 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 6851)
  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..6851
                     /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..3513
                     /label=CRAFi1.3_P2A_FusionREd
     misc_feature    679..2724
                     /label=CRAFi1.3_3KGC_ER2
     CDS             679..2502
                     /codon_start=1
                     /product="Isoform B"
                     /label=hCCR2B
                     /translation="MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLL
                     PPLASLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWAHSAAN
                     EWVFGNAMCKLFTGLAHIGYFGGIFFIILLTIDRYLAIVHAVFALKARTVTFGVVTSVI
                     TWLVAVFASVPGIIFTKCQKEDSVYVCGPYFPRGWNNFHTIMRNILGLVLPLLIMVICY
                     SGILKTLLRCRNGLEEGGNVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIG
                     DGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSK
                     GEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVT
                     TLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVN
                     RIELKGIDFKEDGNILGHKLEYNIDKKRHRAVRVIFTIMIVYFLFWTPYNIVILLNTFQ
                     EFFGLSNCESTSQLDQATQVTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRF
                     CKQCPVFYRETVDGVTSTNTPSTGEQEVSAGL"
     primer_bind     complement(805..850)
                     /label=Y49A_Rv_new
     misc_feature    823..825
                     /label=Y49A
     primer_bind     826..850
                     /label=Y49A_new_Fw
     primer_bind     937..957
                     /label=sequencing primer
     primer_bind     complement(1014..1059)
                     /label=Y120A_Rv_new
     misc_feature    1036..1038
                     /label=Y120A
     primer_bind     1039..1059
                     /label=Y120A_new_Fw
     primer_bind     1300..1317
                     /label=NEW SEQUENCING PRIMER
     misc_feature    1357..2151
                     /label=ICL3
     primer_bind     1384..1425
                     /label=LEGG_GFP_Fw
     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"
     primer_bind     complement(2468..2524)
                     /label=引物 HCCR2b  citrine_KGCX3 Rv
     CDS             2503..2520
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MDELYK"
     CDS             2521..2577
                     /codon_start=1
                     /label=KGC
                     /translation="SRITSEGEYIPLDQIDINV"
     primer_bind     complement(2550..2577)
                     /label=KGC_GG_Rv
     CDS             2578..2637
                     /codon_start=1
                     /label=KGC
                     /translation="KSRITSEGEYIPLDQIDINV"
     primer_bind     complement(2610..2637)
                     /label=KGC_GG_Rv
     CDS             2638..2697
                     /codon_start=1
                     /label=KGC
                     /translation="KSRITSEGEYIPLDQIDINV"
     primer_bind     complement(2670..2703)
                     /label=KGC_GG_Rv
     primer_bind     complement(2698..2724)
                     /label=GGER2_NotI_Rv
     CDS             2704..2724
                     /codon_start=1
                     /label=ER2
                     /translation="FCYENEV"
     CDS             2749..2805
                     /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     2805..2832
                     /label=GGFusionRed_Fw
     primer_bind     complement(2805..2820)
                     /label=KGC_GG_Rv
     CDS             2818..3513
                     /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(3490..3521)
                     /label=FusionRed_GGER2_Rv
     primer_bind     complement(3493..3544)
                     /label=pN1_FusionREd_NotI_Rv
     primer_bind     complement(3514..3544)
                     /label=GGER2_NotI_Rv
     primer_bind     complement(3521..3551)
                     /label=pN1_Not_Rv
     polyA_signal    3639..3760
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3762..3801)
                     /label=polyA_long_Rv
     rep_origin      complement(3767..4222)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        4249..4353
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4355..4712
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4563..4698
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4747..5541
                     /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    5773..5820
                     /label=HSV TK poly(A) signal
                     /note="herpesvirus thymidine kinase polyadenylation signal"
     rep_origin      6149..6737
                     /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 tggccagcct ggtgttcatc
      841 ttcggcttcg tgggcaacat gctggtggtg ctgatcctga tcaactgcaa gaagctgaag
      901 tgcctgaccg acatctacct gctgaacctg gccatcagcg acctgctgtt cctgatcacc
      961 ctgcccctgt gggcccacag cgccgccaac gagtgggtgt tcggcaacgc catgtgcaag
     1021 ctgttcaccg gcctggccca 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 ggattttgtt atgagaatga agtgcgaccg gttcacgtgg gaagtggggc tacaaacttc
     2761 tcactgctga aacaggccgg ggatgtggag gaaaacccag gaccacacgt gactagtatg
     2821 gtgagcgagc tgattaagga gaacatgccc atgaagctgt acatggaggg caccgtgaac
     2881 aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
     2941 atgagaatca aggtcgtcga gggcggccct ctccccttcg ccttcgacat cctggctacc
     3001 agcttcatgt acggcagcag aaccttcatc aagcaccctc cgggcatccc cgacttcttt
     3061 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
     3121 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
     3181 gttagagggg tgaacttccc agccaacggc cctgtgatgc agaagaaaac actcggctgg
     3241 gaggcctcca ccgagacgat gtaccctgct gacggaggac tggaaggcgc atgtgacatg
     3301 gctctgaagc tcgtgggagg aggacacctg atctgcaacc ttgagacaac atacagatcc
     3361 aagaaacccg ctacgaacct caagatgccc ggcgtctaca acgtggacca cagactggaa
     3421 agaatcaagg aggccgacga tgagacatac gtcgagcagc acgaggtggc tgtggccaga
     3481 tactctactg gtggcgctgg tgatggaggt aaataaagcg gccgcgactc tagatcataa
     3541 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     3601 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     3661 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3721 attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt
     3781 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     3841 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     3901 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     3961 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     4021 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     4081 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     4141 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     4201 aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4261 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4321 taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga
     4381 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4441 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     4501 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     4561 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     4621 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     4681 gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg
     4741 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4801 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4861 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4921 gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4981 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     5041 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     5101 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     5161 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     5221 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
     5281 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     5341 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     5401 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     5461 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     5521 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     5581 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5641 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5701 tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     5761 cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc
     5821 ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt
     5881 ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag
     5941 gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat
     6001 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6061 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6121 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6181 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6241 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6301 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6361 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6421 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6481 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6541 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6601 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6661 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6721 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6781 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6841 ccgccatgca t
//