LOCUS       CXCR4-SZ158a-sfG        6571 bp ds-DNA     circular SYN 09-MAR-2021
DEFINITION  Mammalian expression plasmid for RBD of SARS-CoV-2 protein S fused
            with sfGFP.
ACCESSION
VERSION     .
KEYWORDS    pcDNA3-SARS-CoV-2-S-RBD-sfGFP.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 6736)
  AUTHORS   Procko and E
  TITLE     The sequence of human ACE2 is suboptimal for binding the S spike
            protein of SARS coronavirus 2
  JOURNAL   bioRxiv 2020.03.16.994236
REFERENCE   2  (bases 1 to 6736)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2020 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     enhancer        1..380
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     source          1..6571
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        381..584
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /label="CMV promoter"
     primer_bind     535..555
                     /note="Human CMV immediate early promoter, forward primer"
                     /label="CMV-F"
     promoter        629..647
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     primer_bind     629..648
                     /note="T7 promoter, forward primer"
                     /label="T7"
     misc_feature    676..1146
                     /Original_Bases="AAGACCATCATCGCCCTGAGCTACATCTTCTGCCTGGTGTT
                     CGCCACTAATCTTTGTCCGTTCGGTGAGGTTTTTAACGCGACAAGGTTCGCTAGTGTA
                     TATGCTTGGAACCGAAAGAGAATCTCCAATTGCGTAGCTGATTACTCCGTTCTCTATA
                     ACAGTGCGTCCTTTTCAACCTTTAAGTGTTACGGCGTTTCTCCAACGAAGCTGAATGA
                     TCTCTGTTTTACGAACGTGTATGCTGACTCTTTCGTTATACGGGGGGACGAAGTGAGA
                     CAGATAGCACCAGGTCAGACTGGGAAGATAGCGGATTACAACTATAAGTTGCCCGATG
                     ATTTTACGGGGTGCGTAATCGCATGGAACTCAAACAACCTCGACTCCAAAGTAGGTGG
                     TAATTATAATTACTTGTATCGCCTGTTTCGAAAGAGCAATTTGAAGCCTTTTGAGCGG
                     GATATTTCAACCGAAATTTACCAAGCAGGCAGTACGCCATGTAACGGAGTAGAGGGAT
                     TTAATTGCTACTTTCCTCTTCAATCTTATGGCTTTCAACCAACAAACGGAGTGGGGTA
                     TCAACCTTATAGAGTGGTAGTATTGTCCTTTGAGCTCCTCCACGCCCCGGCTACAGTT
                     TGTGGGCCCAAAAAG"
                     /label="AAGACCATCATCGCCCTGAGCTACATCTTCTGCCTGGTGTTCGCCACTAA
                     TCTTTGTCCGTTCGGTGAGGTTTTTAACGCGACAAGGTTCGCTAGTGTATATGCTTGG
                     AACCGAAAGAGAATCTCCAATTGCGTAGCTGATTACTCCGTTCTCTATAACAGTGCGT
                     CCTTTTCAACCTTTAAGTGTTACGGCGTTTCTCCAACGAAGCTGAATGATCTCTGTTT
                     TACGAACGTGTATGCTGACTCTTTCGTTATACGGGGGGACGAAGTGAGACAGATAGCA
                     CCAGGTCAGACTGGGAAGATAGCGGATTACAACTATAAGTTGCCCGATGATTTTACGG
                     GGTGCGTAATCGCATGGAACTCAAACAACCTCGACTCCAAAGTAGGTGGTAATTATAA
                     TTACTTGTATCGCCTGTTTCGAAAGAGCAATTTGAAGCCTTTTGAGCGGGATATTTCA
                     ACCGAAATTTACCAAGCAGGCAGTACGCCATGTAACGGAGTAGAGGGATTTAATTGCT
                     ACTTTCCTCTTCAATCTTATGGCTTTCAACCAACAAACGGAGTGGGGTATCAACCTTA
                     TAGAGTGGTAGTATTGTCCTTTGAGCTCCTCCACGCCCCGGCTACAGTTTGTGGGCCC
                     AAAAAG"
                     /note="Geneious type: Editing History Replacement"
     ORF             676..1146
                     /created_by="User"
                     /label="CXCR4-SZ158a"
     CDS             1177..1884
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /translation="KGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVL
                     LEFVTAAGITHGMDELYK"
                     /label="superfolder GFP"
     primer_bind     1177..1209
                     /Sequence="AAAGGAGAAGAACTTTTCACTGGAGTTGTCCCA"
                     /Hairpin_Tm="36.4"
                     /Tm="67.6"
                     /Self_Dimer_Tm="8.7"
                     /%GC="42.4"
                     /created_by="primer3"
                     /label
     primer_bind     1188..1209
                     /created_by="primer3"
                     /%GC="45.5"
                     /Tm="59.8"
                     /Hairpin_Tm="43.2"
                     /Self_Dimer_Tm="None"
                     /Sequence="ACTTTTCACTGGAGTTGTCCCA"
                     /annotation_group="67136838748800207ade663-20b5-4333-b73c-
                     c72dade1680eurn:sequence:genbank:."
                     /label="1,353 F"
     primer_bind     complement(1201..1229)
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
                     /label="GFP-R"
     primer_bind     1824..1844
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
                     /label="GFP-F"
     primer_bind     complement(1928..1945)
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
                     /label="BGH-rev"
     polyA_signal    1934..2158
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      2204..2632
                     /direction="RIGHT"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     primer_bind     complement(2291..2310)
                     /note="F1 origin, reverse primer"
                     /label="F1ori-R"
     primer_bind     2501..2522
                     /note="F1 origin, forward primer"
                     /label="F1ori-F"
     primer_bind     complement(2641..2661)
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
                     /label="pBABE 3'"
     promoter        2646..2975
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     rep_origin      2826..2961
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     2888..2907
                     /note="SV40 promoter/origin, forward primer"
                     /label="SV40pro-F"
     CDS             3042..3836
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="confers resistance to neomycin, kanamycin, and G418
                      (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
                     QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
                     /label="NeoR/KanR"
     primer_bind     complement(3096..3115)
                     /note="Neomycin resistance gene, reverse primer"
                     /label="Neo-R"
     primer_bind     3706..3725
                     /note="Neomycin resistance gene, forward primer"
                     /label="Neo-F"
     polyA_signal    4010..4131
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(4047..4066)
                     /note="SV40 polyA, reverse primer"
                     /label="SV40pA-R"
     primer_bind     4101..4120
                     /note="SV40 polyA terminator, reverse primer"
                     /label="EBV-rev"
     primer_bind     complement(4180..4196)
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /label="M13 rev"
     primer_bind     complement(4180..4196)
                     /note="In lacZ gene. Also called M13-rev"
                     /label="M13 Reverse"
     primer_bind     complement(4193..4215)
                     /note="In lacZ gene"
                     /label="M13/pUC Reverse"
     protein_bind    4204..4220
                     /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)."
                     /label="lac operator"
     promoter        complement(4228..4258)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    4273..4294
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /label="CAP binding site"
     primer_bind     complement(4411..4428)
                     /note="L4440 vector, forward primer"
                     /label="L4440"
     rep_origin      complement(4582..5170)
                     /direction="LEFT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     primer_bind     complement(4662..4681)
                     /note="pBR322 origin, forward primer"
                     /label="pBR322ori-F"
     CDS             complement(5341..6201)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /label="AmpR"
     primer_bind     5964..5983
                     /note="Ampicillin resistance gene, reverse primer"
                     /label="Amp-R"
     promoter        complement(6202..6306)
                     /gene="bla"
                     /label="AmpR promoter"
     primer_bind     complement(6381..6400)
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /label="pRS-marker"
ORIGIN      
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgcca ccatggaggg gatcagtata tacacttcag ataactacac cgaggaaatg
      721 ggctcagggg actatgactc catgaaggaa ccctgtttcc gtgaagaaaa tgctaatttc
      781 aataaaatct tcctgcccac catctactcc atcatcttct taactctggc tttcttcgcc
      841 tgttggctgc cttactacat tgggatcagc atcgactcct tcatcctcct ggaaatcatc
      901 aagcaagggt gtgagtttga gaacactgtg cacaagtgga tttccatcac cgaggcccta
      961 gctttcttcc actgttgtct gaaccccatc ctctatgctt tccttggagc caaatttaaa
     1021 acctctgccc agcacgcact cacctctgtg agcagagggt ccagcctcaa gatcctctcc
     1081 aaaggaaagc gaggtggaca ttcatctgtt tccactgagt ctgagtcttc aagttttcac
     1141 tccagcggat ccggtgggtc cggtagtggt ggttccaaag gagaagaact tttcactgga
     1201 gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtccgt
     1261 ggagagggtg aaggtgatgc tacaaacgga aaactcaccc ttaaatttat ttgcactact
     1321 ggaaaactac ctgttccatg gccaacactt gtcactactc tgacctatgg tgttcaatgc
     1381 ttttcccgtt atccggatca catgaaacgg catgactttt tcaagagtgc catgcccgaa
     1441 ggttatgtac aggaacgcac tatatctttc aaagatgacg ggacctacaa gacgcgtgct
     1501 gaagtcaagt ttgaaggtga tacccttgtt aatcgtatcg agttaaaagg tattgatttt
     1561 aaagaagatg gaaacattct cggacacaaa ctggagtaca actttaactc acacaatgta
     1621 tacatcacgg cagacaaaca aaagaatgga atcaaagcta acttcaaaat tcgccacaac
     1681 gttgaagatg gttccgttca actagcagac cattatcaac aaaatactcc aattggcgat
     1741 ggccctgtcc ttttaccaga caaccattac ctgtcgacac aatctgtcct ttcgaaagat
     1801 cccaacgaaa agcgtgacca catggtcctt cttgagtttg taactgctgc tgggattaca
     1861 catggcatgg atgagctcta caaatgataa ctcgagtcta gagggcccgt ttaaacccgc
     1921 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     1981 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2041 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2101 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     2161 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     2221 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2281 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2341 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2401 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2461 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2521 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     2581 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt
     2641 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     2701 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     2761 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     2821 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     2881 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt
     2941 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca
     3001 ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3061 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3121 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     3181 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc
     3241 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     3301 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     3361 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     3421 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     3481 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     3541 cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga
     3601 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     3661 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     3721 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     3781 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     3841 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     3901 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     3961 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat
     4021 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4081 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4141 tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4201 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4261 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4321 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4381 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4441 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4501 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4561 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4621 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4681 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4741 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4801 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4861 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4921 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4981 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5041 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5101 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5161 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5221 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5281 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5341 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5401 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5461 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5521 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5581 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5641 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5701 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5761 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5821 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5881 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5941 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6001 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6061 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6121 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6181 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6241 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6301 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat
     6361 cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg
     6421 ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca
     6481 aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg
     6541 cttcgcgatg tacgggccag atatacgcgt t
//
