LOCUS       pENTR1a-iAspSnFR        4669 bp DNA     circular SYN 21-SEP-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 4669)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported May 29, 2023 from SnapGene 6.2.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4669
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     terminator      103..189
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      281..308
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     328..351
                     /label=pENTR1a_FW_insert
     protein_bind    358..457
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    complement(358..441)
                     /label=attL2
     primer_bind     complement(494..515)
                     /label=pENTR1a_GA_RV
     primer_bind     499..534
                     /label=Asp-sens-RFP_noMTS_FW
     misc_feature    516..521
                     /label=Kozak
     CDS             522..533
                     /codon_start=1
                     /label=MCS
                     /note="MCS"
                     /translation="MGRS"
     primer_bind     525..542
                     /label=Asp-sens-RFP_MTS_FW
     CDS             534..1280
                     /codon_start=1
                     /label=ybeJ1-253
                     /note="ybeJ1-253"
                     /translation="AAGSTLDKIAKNGVIVVGHRKSAVPFSYYDSQHKVVGYSQEYSNA
                     IVEAVKKKLNKPDLQVKLIPITPQNRIPLLQNGTYDFECGSTTNNVERQKQAAFSDTIF
                     VVGTRLLTKKGGDIKDFANLKDKAVVVTSGTTSEVLLNKLNEEQKMNMRIISAKDHGDS
                     FRTLESGRAVAFMMDDVLLAGERAKAKKPDDWEIVGKPQSQEAYGCMLRKDDPQFKKLM
                     DDTIAQVQTSGEAEKWFDKWFKNPILV"
     misc_feature    594..596
                     /label=E25K (akaE23K, E25D in iGluSnFR3)
     misc_feature    600..602
                     /label=S27A (Asp specificity)
     misc_feature    624..626
                     /label=N35S (iGluSnFR3 mutation)
     misc_feature    630..632
                     /label=Q37H (iGluSnFR3 mutations)
     misc_feature    654..656
                     /label=D45E (iGluSnFR3 mutation)
     misc_feature    735..737
                     /label=S72P (Asp specificity)
     misc_feature    771..773
                     /label=F84Y (iGluSnFR3 mutation)
     misc_feature    1071..1073
                     /label=A184V
     misc_feature    1113..1115
                     /label=N198D (iGluSnFR3 mutation)
     CDS             1281..1556
                     /codon_start=1
                     /label=cpSF Venus
                     /translation="SHIVYITADKQKNGIKANFKIHHNVEDGSVQLADHYQQNTPIGDG
                     PVLLPDNHYLSYQSVLSKDPNEKRDHMVLLEFVTATTNSLGMDELYK"
     CDS             1557..1574
                     /codon_start=1
                     /label=cpsfGFP
                     /note="cpsfGFP"
                     /translation="GGTGGS"
     CDS             1575..2012
                     /codon_start=1
                     /label=cpSF Venus
                     /note="cpSF Venus"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     LICTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKLEGDTLVNRIELKGIDFKEDGNILGHKLEYNFN"
     CDS             2013..2090
                     /codon_start=1
                     /label=YbeJ254-280
                     /note="YbeJ254-280"
                     /translation="NPLNMNFELSDEMKALFKEPNDKALK"
     CDS             2091..2096
                     /codon_start=1
                     /label=MCS
                     /translation="LQ"
     CDS             2097..2141
                     /codon_start=1
                     /label=linker
                     /note="linker"
                     /translation="STGSGNAVGQDTQER"
     CDS             2142..2852
                     /codon_start=1
                     /label=mRuby3
                     /note="mRuby3"
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     misc_feature    2250..2252
                     /label=N33R
                     /note="N33R"
     misc_feature    2259..2261
                     /label=M36E
                     /note="M36E"
     misc_feature    2265..2267
                     /label=T38V
                     /note="T38V"
     misc_feature    2373..2375
                     /label=K74A
                     /note="K74A"
     misc_feature    2376..2378
                     /label=G75D
                     /note="G75D"
     misc_feature    2466..2468
                     /label=M105T
                     /note="M105T"
     misc_feature    2493..2495
                     /label=C114E
                     /note="C114E"
     misc_feature    2505..2507
                     /label=H118N
                     /note="H118N"
     misc_feature    2511..2513
                     /label=Q120K
                     /note="Q120K"
     misc_feature    2628..2630
                     /label=H159D
                     /note="H159D"
     misc_feature    2631..2633
                     /label=M160I
                     /note="M160I"
     misc_feature    2664..2666
                     /label=S171H
                     /note="S171H"
     misc_feature    2670..2672
                     /label=S173N
                     /note="S173N"
     misc_feature    2727..2729
                     /label=I192V
                     /note="I192V"
     misc_feature    2757..2759
                     /label=L202I
                     /note="L202I"
     misc_feature    2778..2780
                     /label=M209T
                     /note="M209T"
     misc_feature    2781..2783
                     /label=F210Y
                     /note="F210Y"
     misc_feature    2799..2801
                     /label=H216V
                     /note="H216V"
     misc_feature    2814..2816
                     /label=F221Y
                     /note="F221Y"
     misc_feature    2817..2819
                     /label=A222S
                     /note="A222S"
     misc_feature    2820..2822
                     /label=G223N
                     /note="G223N"
     CDS             2856..2873
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2870..2911)
                     /label=Asp-sens-RFP_RV
     primer_bind     2894..2914
                     /label=pENTR1a_GA_FW
     protein_bind    complement(2898..2997)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2989..3008)
                     /label=pENTR1a_RV_insert
     gene            3120..3929
                     /gene="KanR2 (variant)"
                     /label=KanR2 (variant)
     CDS             3120..3929
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVSLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      4003..4622
                     /label=pBR322_origin
                     /note="/translation=MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIY
                     RLYGK 
                     PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK 
                     TAFQVLEEYPDSGENIVDALAVSLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVD 
                     ASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRV 
                     GIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF*"
     rep_origin      4019..4607
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagca tggatctcgg
       61 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
      121 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
      181 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
      241 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
      301 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagct cgggccccaa
      361 ataatgattt tattttgact gatagtgacc tgttcgttgc aacaaattga taagcaatgc
      421 ttttttataa tgccaacttt gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac
      481 tggatccggt accgaattcg cttactaaaa gccaggccac catgggcaga tctgccgcag
      541 gcagcacgct ggacaaaatc gccaaaaacg gtgtgattgt cgtcggtcac cgtaaatctg
      601 ccgtgccttt ctcttattac gacagtcagc acaaagtggt gggttactcg caggaatact
      661 ccaacgccat tgttgaagca gtgaaaaaga aactcaacaa accggacttg caggtaaaac
      721 tgattccgat taccccacaa aaccgtattc cactgctgca aaacggcact tacgattttg
      781 aatgtggttc taccaccaac aacgtcgaac gccaaaaaca ggcggctttc tctgacacta
      841 ttttcgtggt cggtacgcgc ctgttgacca aaaagggtgg cgatatcaaa gattttgcca
      901 acctgaaaga caaagccgta gtcgtcactt ccggcactac ctctgaagtt ttgctcaaca
      961 aactgaatga agagcaaaaa atgaatatgc gcatcatcag cgccaaagat cacggtgact
     1021 ctttccgcac cctggaaagc ggtcgtgccg ttgcctttat gatggatgac gtgctgctgg
     1081 ccggtgaacg tgcgaaagcg aagaaaccag acgactggga aatcgtcggc aagccgcagt
     1141 ctcaggaggc ctacggttgt atgttgcgta aagatgatcc gcagttcaaa aagctgatgg
     1201 atgacaccat cgctcaggtg cagacctccg gtgaagcgga aaaatggttt gataagtggt
     1261 tcaaaaatcc aattctggtg agccacatcg tctatatcac cgccgacaag cagaagaacg
     1321 gcatcaaggc gaacttcaag atccaccaca acgtggagga cggcagcgtg cagctcgccg
     1381 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1441 acctgagcta tcagtccgtg ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     1501 tgctggagtt cgtgaccgcc accacgaact ctctcggcat ggacgagctg tacaagggcg
     1561 gtaccggagg gagcatgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1621 tcgagctgga cggcgacgta aacggccaca agttcagcgt gcgcggcgag ggcgagggcg
     1681 atgccaccaa cggcaagcta accctgaagc tcatctgcac caccggcaag ctgcccgtgc
     1741 cctggcccac cctcgtgacc accctgggct acggcgtgca gtgcttcgcc cgctaccccg
     1801 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1861 gcaccatcag cttcaaggac gacggaacct acaagacccg cgccgaggtg aagttagagg
     1921 gcgacaccct ggtgaaccgc atcgagctaa agggcatcga cttcaaggag gacggcaaca
     1981 tcctggggca caagctggag tacaacttta acaaccccct gaacatgaat ttcgaactgt
     2041 cagacgaaat gaaagcactg ttcaaagaac cgaatgacaa ggcactgaaa ctgcagtcta
     2101 ccggctccgg aaatgctgtg ggccaggaca cgcaggagcg tatggtgtct aagggcgaag
     2161 agctgatcaa ggaaaatatg cgtatgaagg tggtcatgga aggttcggtc aacggccacc
     2221 aattcaaatg cacaggtgaa ggagaaggca ggccgtacga gggagtgcaa accatgagga
     2281 tcaaagtcat cgagggagga cccctgccat ttgcctttga cattcttgcc acgtcgttca
     2341 tgtatggcag ccgtactttt atcaagtacc cggcagacat tcctgatttc tttaaacagt
     2401 cctttcctga gggttttact tgggaaagag ttacgagata cgaagatggt ggagtcgtca
     2461 ccgtcacgca ggacaccagc cttgaggatg gcgaactcgt ttacaacgtc aaagtcagag
     2521 gggtaaactt tccctccaat ggtcccgtga tgcagaagaa gaccaagggt tgggagccta
     2581 atacagagat gatgtatcca gcagatggtg gtctgagggg atacactgat attgcactga
     2641 aagttgatgg tggtggccat ctgcattgca atttcgtaac aacttacagg tcaaaaaaga
     2701 ccgtcgggaa catcaagatg cccggtgtcc atgccgttga tcaccgcctg gaaaggatag
     2761 aggaaagtga caatgaaacg tacgtagtac aacgcgaagt cgcagttgcc aagtactcca
     2821 atcttggtgg tggtatggat gaactttata agtcccatca ccatcaccac cattaagaat
     2881 tcgaagctta tcgctagacc cagctttctt gtacaaagtt ggcattataa gaaagcattg
     2941 cttatcaatt tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgcca
     3001 tccagctgca gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa
     3061 atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta
     3121 tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt
     3181 tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct
     3241 tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca
     3301 atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga
     3361 ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg
     3421 gaaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg
     3481 cgctggcagt gtccctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca
     3541 gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg
     3601 cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc
     3661 ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata
     3721 accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg
     3781 cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat
     3841 tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt
     3901 ttcatttgat gctcgatgag tttttctaat cagaattggt taattggttg taacattatt
     3961 cagattgggc cccgttccac tgagcgtcag acccggtaga aaagatcaaa ggatcttctt
     4021 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4081 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4141 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4201 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4261 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4321 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4381 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4441 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4501 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4561 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4621 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtt
//
