LOCUS       Exported                7953 bp DNA     circular SYN 02-FEB-2022
DEFINITION  Expresses Kir4.1 fused with BioID2 at the N-terminus in astrocytes.
ACCESSION   .
VERSION     .
KEYWORDS    pZac2.1 gfaABC1D-BioID2-BioID2-Kir4.1-HA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7953)
  TITLE     Bacterial expression plasmids for production of protein-fused BioID2
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7953)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Feb 2, 2022 from SnapGene 5.3.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
FEATURES             Location/Qualifiers
     source          1..7953
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /label=lac operator
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   259..388
                     /label=AAV2 ITR
     primer_bind     complement(270..281)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     331..342
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(794..805)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     intron          1239..1371
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1354..1373
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1416..1434
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1461..2159
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="MFKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKW
                     LSQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKV
                     SGVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISE
                     NLKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKE
                     ILSGEFSLRRS"
     misc_feature    2160..2243
                     /label=Linker
     CDS             2244..2942
                     /codon_start=1
                     /product="promiscuous R40G mutant of a biotin protein 
                     ligase from Aquifex aeolicus (Kim et al., 2016)"
                     /label=BioID2
                     /translation="MFKNLIWLKEVDSTQERLKEWNVSYGTALVADRQTKGRGGLGRKW
                     LSQEGGLYFSFLLNPKEFENLLQLPLVLGLSVSEALEEITEIPFSLKWPNDVYFQEKKV
                     SGVLCELSKDKLIVGIGINVNQREIPEEIKDRATTLYEITGKDWDRKEVLLKVLKRISE
                     NLKKFKEKSFKEFKGKIESKMLYLGEEVKLLGEGKITGKLVGLSEKGGALILTEEGIKE
                     ILSGEFSLRRS"
     misc_feature    2943..3134
                     /label=Linker
     misc_feature    3135..4271
                     /label=Kcnj10 (Kir4.1) cDNA
     CDS             4275..4301
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    4326..4914
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4379..4399)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4797..4808)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4978..5099
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4989..5008)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5043..5062
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5261..5390
                     /label=AAV2 ITR
     primer_bind     complement(5307..5318)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5368..5379
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5409..5426)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5409..5425)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5418..5440)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5567..6022
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5654..5673)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5864..5885
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6048..6152
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6153..7013
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6371..6390)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7184..7772
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7673..7692
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7926..7943
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta
      241 attaaggcct taattaggct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
      301 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
      361 gtggccaact ccatcactag gggttccttg tagttaatga ttaacccgcc atgctactta
      421 tctacgtagc catgctctag gaagatctaa catatcctgg tgtggagtag gggacgctgc
      481 tctgacagag gctcgggggc ctgagctggc tctgtgagct ggggaggagg cagacagcca
      541 ggccttgtct gcaagcagac ctggcagcat tgggctggcc gccccccagg gcctcctctt
      601 catgcccagt gaatgactca ccttggcaca gacacaatgt tcggggtggg cacagtgcct
      661 gcttcccgcc gcaccccagc ccccctcaaa tgccttccga gaagcccatt gagcaggggg
      721 cttgcattgc accccagcct gacagcctgg catcttggga taaaagcagc acagccccct
      781 aggggctgcc cttgctgtgt ggcgccaccg gcggtggaga acaaggctct attcagcctg
      841 tgcccaggaa aggggatcag gggatgccca ggcatggaca gtgggtggca gggggggaga
      901 ggagggctgt ctgcttccca gaagtccaag gacacaaatg ggtgagggga gagctctccc
      961 catagctggg ctgcggccca accccacccc ctcaggctat gccagggggt gttgccaggg
     1021 gcacccgggc atcgccagtc tagcccactc cttcataaag ccctcgcatc ccaggagcga
     1081 gcagagccag agcaggttgg agaggagacg catcacctcc gctgctcgca agctttattg
     1141 cggtagttta tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga
     1201 acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
     1261 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
     1321 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
     1381 cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta taggctagcc
     1441 tcgagctcgg atccaccacc atgttcaaaa atcttatttg gctgaaggaa gttgatagca
     1501 cccaggagcg gcttaaggaa tggaatgtct cctatggcac tgcactcgtc gccgatagac
     1561 agaccaaagg tagaggcggt ctcggacgga agtggttgag tcaggagggc ggtctttact
     1621 tctctttctt gttgaatcca aaagagtttg aaaatttgtt gcaacttcca ctggtcttgg
     1681 gactctccgt gagcgaggca ctcgaagaga ttacagagat accatttagt ctgaagtggc
     1741 ccaacgacgt ttattttcag gagaaaaagg tttcaggggt cctttgtgag ctcagtaagg
     1801 acaagctgat agtgggaatc ggtataaacg tgaatcaaag agagatccct gaagagatca
     1861 aggatcgggc cacaacactt tatgaaatca ctggaaagga ttgggacaga aaggaggtct
     1921 tgctgaaggt tctcaagcga atttccgaaa atctcaagaa atttaaggaa aaatccttta
     1981 aagagttcaa aggaaaaatt gagtcaaaga tgttgtacct tggagaggag gttaaactct
     2041 tgggagaagg caagattacc gggaaacttg tgggcctgag tgaaaagggc ggtgctttga
     2101 tacttacaga ggaaggcata aaggagatac tgtccggcga atttagcttg cgacggtcaa
     2161 gcttaggtgg aggaggttct ggaggcggtg gaagtggtgg cggaggtagc ggtggaggag
     2221 gttctatggt gtctaaggaa ttcatgttca aaaatcttat ttggctgaag gaagttgata
     2281 gcacccagga gcggcttaag gaatggaatg tctcctatgg cactgcactc gtcgccgata
     2341 gacagaccaa aggtagaggc ggtctcggac ggaagtggtt gagtcaggag ggcggtcttt
     2401 acttctcttt cttgttgaat ccaaaagagt ttgaaaattt gttgcaactt ccactggtct
     2461 tgggactctc cgtgagcgag gcactcgaag agattacaga gataccattt agtctgaagt
     2521 ggcccaacga cgtttatttt caggagaaaa aggtttcagg ggtcctttgt gagctcagta
     2581 aggacaagct gatagtggga atcggtataa acgtgaatca aagagagatc cctgaagaga
     2641 tcaaggatcg ggccacaaca ctttatgaaa tcactggaaa ggattgggac agaaaggagg
     2701 tcttgctgaa ggttctcaag cgaatttccg aaaatctcaa gaaatttaag gaaaaatcct
     2761 ttaaagagtt caaaggaaaa attgagtcaa agatgttgta ccttggagag gaggttaaac
     2821 tcttgggaga aggcaagatt accgggaaac ttgtgggcct gagtgaaaag ggcggtgctt
     2881 tgatacttac agaggaaggc ataaaggaga tactgtccgg cgaatttagc ttgcgacggt
     2941 cagctagcga ctgccattat cccccgccgg gtggaggcgg gtcgggtggc ggaggtagcg
     3001 gaggcggtgg aagtggaggc ggtggaagtg gtggaggagg ttctggtggc ggaggtagcg
     3061 gtggcggagg tagcggaggc ggtggaagtg gtggcggagg tagcggaggc ggtggatcgg
     3121 tcgacacgat gttatcgaat tctgttgcca aggtctatta cagccagacg acgcagacag
     3181 agagccggcc cctagtggcc ccaggaatac gtcggaggag ggtcctgaca aaagatggcc
     3241 ggagcaacgt gagaatggag catattgctg acaagcgttt cctctacctc aaggatctat
     3301 ggacgacctt cattgacatg cagtggcgct acaagcttct gctcttctcg gcaacctttg
     3361 caggcacttg gttcctcttt ggcgtggtgt ggtatctggt cgctgtggcc cacggggacc
     3421 tgttggagct gggacctcct gccaaccaca cgccctgtgt ggtgcaggtg cacacactta
     3481 ctggggcctt cctcttctcc ctcgaatccc agaccaccat tggctatggc ttccgctaca
     3541 tcagcgagga atgccctctg gccattgtgc ttctcattgc acagctcgtg ctcaccacca
     3601 ttctggaaat cttcatcacc ggaaccttcc ttgcaaagat tgcccggccg aagaagaggg
     3661 ctgagacgat ccgtttcagc cagcatgcgg ttgtggctta ccacaacggg aagctttgcc
     3721 tcatgatccg ggtggccaac atgcgtaaga gtctcctcat tgggtgccag gtgacaggca
     3781 aactgcttca aacccaccag acaaaggagg gtgagaatat tcggctcaac caggtcaatg
     3841 tgactttcca agtagacaca gcctctgata gcccctttct cattctaccc ctgactttct
     3901 accatgtggt agatgagacc agccccttga aagatctccc cctccgcagc ggggagggtg
     3961 acttcgagct cgtgctgatc ctaagtggga cggtggagtc caccagcgcc acctgtcaag
     4021 ttcgcacttc ctatctaccg gaggagatcc tctggggcta cgagttcaca cctgctatct
     4081 cactgtcagc cagtggcaaa tacgtggctg acttcagcct ttttgaccag gttgtgaaag
     4141 tggcgtcccc cggtggtctc cgagatagca ccgtacgtta tggagaccca gaaaagctca
     4201 agttggagga gtcattaaga gagcaagctg aaaaggaagg cagtgccctt agtgtgcgca
     4261 ttagtaacgt cgcttaccct tacgatgtac cggattacgc atagggatcc aattcgctta
     4321 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     4381 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     4441 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     4501 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     4561 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     4621 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     4681 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     4741 tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     4801 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     4861 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     4921 accggaattc tgcagatatc tagagtcgac ccgggcggcc gcttcgagca gacatgataa
     4981 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     5041 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     5101 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt
     5161 aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatcttcct agagcatggc
     5221 tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag
     5281 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     5341 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ccttaattaa
     5401 cctaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     5461 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     5521 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     5581 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     5641 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     5701 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5761 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     5821 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5881 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5941 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     6001 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     6061 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     6121 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     6181 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     6241 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     6301 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     6361 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     6421 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     6481 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     6541 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     6601 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     6661 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     6721 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     6781 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6841 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6901 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6961 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     7021 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     7081 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     7141 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     7201 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     7261 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     7321 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     7381 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     7441 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     7501 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     7561 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     7621 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     7681 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7741 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7801 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7861 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     7921 agcgcagcga gtcagtgagc gaggaagcgg aag
//
