LOCUS       PC2328                  5974 bp ds-DNA     circular SYN 28-AUG-2024
DEFINITION  pcDNA3-Esp3I-GSGSG-OTUB1(Catalytic Domain) .
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..5974
                     /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"
     binding_site    667..672
                     /created_by="User"
                     /label
     primer_bind     complement(667..711)
                     /Sequence="GCCGCTGCCGCTGCCGGAGACGCATTAATCGTCTCCCATGGTGGC"
                     /Tm="76.7"
                     /%GC="66.7"
                     /Hairpin_Tm="55.0"
                     /Self_Dimer_Tm="36.8"
                     /created_by="vivia"
                     /label="bb1913_GSGSG_GibR"
     primer_bind     complement(667..686)
                     /%GC="55.0"
                     /Hairpin_Tm="35.8"
                     /Self_Dimer_Tm="11.7"
                     /Tm="59.5"
                     /annotation_group="2823628314927500a7a543e8-5199-4713-a2bb
                     -5b9765da9f1e"
                     /Sequence="TAATCGTCTCCCATGGTGGC"
                     /created_by="primer3"
                     /#_Local_Off-target_Sites=0
                     /Local_Off-target="pcDNA3-Esp3I-GAPGSG-UCHL1"
                     /label="686 R"
     ORF             673..>675
                     /created_by="User"
                     /label
     misc_feature    676..1278
                     /Original_Bases
                     /label
                     /note="Geneious type: Editing History Insertion"
     intron          676..696
                     /created_by="User"
                     /label="Esp3I Cassette"
     overhang        676..696
                     /created_by="User"
                     /label
     source          <676..>696
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     modified_base   complement(677..682)
                     /created_by="User"
                     /label
     misc_feature    complement(677..682)
                     /created_by="User"
                     /label
                     /note="Geneious type: Inserted sequence"
     misc_feature    690..695
                     /created_by="User"
                     /label
                     /note="Geneious type: Inserted sequence"
     polylinker      697..711
                     /created_by="vivia"
                     /label
     source          <697..>711
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          join(<697..>711,<697..>711)
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    712..1287
                     /created_by="lauren_hong11"
                     /label="OTUB1 Cat Domain"
     ORF             <712..1287
                     /created_by="lauren_hong11"
                     /label="OTUB1"
     source          <712..>1287
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          join(<712..>1287,<712..>1287)
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     712..771
                     /created_by="vivia"
                     /Sequence="TCGTACATCCGCAAGACCAGGCCTGACGGCAACTGTTTCTATCGGGC
                     TTTCGGATTCTCC"
                     /Tm="74.8"
                     /%GC="55.0"
                     /Hairpin_Tm="48.4"
                     /Self_Dimer_Tm="28.5"
                     /modified_by="vivia"
                     /label="2028_OTUB-CatDom_KLDF"
     motif           736..738
                     /created_by="laurenhong11"
                     /label="D88"
     motif           745..747
                     /created_by="laurenhong11"
                     /label="C91"
     primer_bind     751..771
                     /%GC="52.4"
                     /Hairpin_Tm="36.9"
                     /Self_Dimer_Tm="None"
                     /Tm="59.7"
                     /annotation_group="4522509098541700ab847bf5-0fd9-4393-98d1
                     -7079fac78304"
                     /Sequence="TATCGGGCTTTCGGATTCTCC"
                     /created_by="primer3"
                     /#_Local_Off-target_Sites=0
                     /Local_Off-target="PC2028 Copy"
                     /label="751 F"
     primer_bind     complement(1258..1317)
                     /created_by="vivia"
                     /Sequence="TTAAACGGGCCCTCTAGACTCGAGTTATCATTTGTAGAGGATATCGT
                     AGTGTCCAGGCCG"
                     /Tm="70.6"
                     /%GC="48.3"
                     /Hairpin_Tm="38.1"
                     /Self_Dimer_Tm="27.0"
                     /modified_by="vivia"
                     /label="1954_OTUB1_GibR"
     primer_bind     complement(1258..1277)
                     /%GC="55.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="59.0"
                     /annotation_group="28245587479473008f4137aa-ba96-416f-a645
                     -740deacb7aa7"
                     /Sequence="ATATCGTAGTGTCCAGGCCG"
                     /created_by="primer3"
                     /#_Local_Off-target_Sites=0
                     /Local_Off-target="pcDNA3-Esp3I-GAPGSG-OTUB1"
                     /label="1,514 R"
     motif           1267..1269
                     /created_by="laurenhong11"
                     /label="H265"
     primer_bind     1288..1311
                     /created_by="vivia"
                     /Sequence="TGATAACTCGAGTCTAGAGGGCCC"
                     /Tm="62.8"
                     /%GC="54.2"
                     /Hairpin_Tm="39.9"
                     /Self_Dimer_Tm="20.3"
                     /modified_by="vivia"
                     /label="bb1913_GibF"
     primer_bind     complement(1331..1348)
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
                     /label="BGH-rev"
     polyA_signal    1337..1561
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      1607..2035
                     /direction="RIGHT"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     primer_bind     complement(1694..1713)
                     /note="F1 origin, reverse primer"
                     /label="F1ori-R"
     primer_bind     1904..1925
                     /note="F1 origin, forward primer"
                     /label="F1ori-F"
     primer_bind     complement(2044..2064)
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
                     /label="pBABE 3'"
     promoter        2049..2378
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     rep_origin      2229..2364
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     2291..2310
                     /note="SV40 promoter/origin, forward primer"
                     /label="SV40pro-F"
     CDS             2445..3239
                     /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(2499..2518)
                     /note="Neomycin resistance gene, reverse primer"
                     /label="Neo-R"
     primer_bind     3109..3128
                     /note="Neomycin resistance gene, forward primer"
                     /label="Neo-F"
     polyA_signal    3413..3534
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(3450..3469)
                     /note="SV40 polyA, reverse primer"
                     /label="SV40pA-R"
     primer_bind     3504..3523
                     /note="SV40 polyA terminator, reverse primer"
                     /label="EBV-rev"
     primer_bind     complement(3583..3599)
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /label="M13 rev"
     primer_bind     complement(3583..3599)
                     /note="In lacZ gene. Also called M13-rev"
                     /label="M13 Reverse"
     primer_bind     complement(3596..3618)
                     /note="In lacZ gene"
                     /label="M13/pUC Reverse"
     protein_bind    3607..3623
                     /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(3631..3661)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    3676..3697
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the
                     presenceof cAMP."
                     /label="CAP binding site"
     primer_bind     complement(3814..3831)
                     /note="L4440 vector, forward primer"
                     /label="L4440"
     rep_origin      complement(3985..4573)
                     /direction="LEFT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     primer_bind     complement(4065..4084)
                     /note="pBR322 origin, forward primer"
                     /label="pBR322ori-F"
     CDS             complement(4744..5604)
                     /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     5367..5386
                     /note="Ampicillin resistance gene, reverse primer"
                     /label="Amp-R"
     promoter        complement(5605..5709)
                     /gene="bla"
                     /label="AmpR promoter"
     primer_bind     complement(5784..5803)
                     /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 ccatgggaga cgattaatgc gtctccggca gcggcagcgg ctcgtacatc
      721 cgcaagacca ggcctgacgg caactgtttc tatcgggctt tcggattctc ccacttggag
      781 gcactgctgg atgacagcaa ggagttgcag cggttcaagg ctgtgtctgc caagagcaag
      841 gaagacctgg tgtcccaggg cttcactgaa ttcacaattg aggatttcca caacacgttc
      901 atggacctga ttgagcaggt ggagaagcag acctctgtcg ccgacctgct ggcctccttc
      961 aatgaccaga gcacctccga ctaccttgtg gtctacctgc ggctgctcac ctcgggctac
     1021 ctgcagcgcg agagcaagtt cttcgagcac ttcatcgagg gtggacggac tgtcaaggag
     1081 ttctgccagc aggaggtgga gcccatgtgc aaggagagcg accacatcca catcattgcg
     1141 ctggcccagg ccctcagcgt gtccatccag gtggagtaca tggaccgcgg cgagggcggc
     1201 accaccaatc cgcacatctt ccctgagggc tccgagccca aggtctacct tctctaccgg
     1261 cctggacact acgatatcct ctacaaatga taactcgagt ctagagggcc cgtttaaacc
     1321 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     1381 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     1441 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     1501 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     1561 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     1621 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1681 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1741 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1801 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1861 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1921 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1981 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     2041 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     2101 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     2161 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     2221 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     2281 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     2341 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     2401 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     2461 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     2521 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     2581 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     2641 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     2701 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     2761 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     2821 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     2881 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     2941 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     3001 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     3061 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     3121 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     3181 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     3241 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     3301 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     3361 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     3421 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     3481 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     3541 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     3601 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3661 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3721 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3781 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3841 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3901 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3961 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4021 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4081 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4141 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4201 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4261 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4321 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4381 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4441 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4501 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4561 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4621 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4681 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4741 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4801 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4861 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4921 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4981 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5041 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5101 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5161 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5221 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5281 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5341 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5401 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5461 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5521 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5581 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5641 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5701 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
     5761 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     5821 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
     5881 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
     5941 ctgcttcgcg atgtacgggc cagatatacg cgtt
//
