LOCUS       Exported                8449 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  donor vector for knock-in gene editing at human DNM1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    pRS DV DNM1-EGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8449)
  AUTHORS   Srinivasan S, Burckhardt CJ, Bhave M, Chen Z, Chen PH, Wang X, 
            Danuser G, Schmid SL
  TITLE     A noncanonical role for dynamin-1 in regulating early stages of 
            clathrin-mediated endocytosis in non-neuronal cells.
  JOURNAL   PLoS Biol. 2018 Apr 18;16(4):e2005377. doi: 
            10.1371/journal.pbio.2005377. eCollection 2018 Apr.
  PUBMED    29668686
REFERENCE   2  (bases 1 to 8449)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8449
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     27..46
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        27..45
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1088..1804
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1130..1151)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1391..1410)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1738..1759
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1927..2048
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1964..1983)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2018..2037
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3007..3027)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3007..3025)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3046..3062)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3046..3062)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3059..3081)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3070..3086
                     /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)."
     promoter        complement(3094..3124)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3277..3294)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3448..4036)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3528..3547)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4207..5067)
                     /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     4830..4849
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5068..5172)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5199..6541
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(6162..6209)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     6582..6600
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6638..6660)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6760..6779
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        6796..7076
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             7077..7751
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(7850..8305)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7987..8008)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8199..8218
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8435..8
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8449..17
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtacca
       61 cgcagtgtag gagccgcgtc tgaaaagcca caccagctca cagtcccagc agggcccggt
      121 ggggcggcca gggcggcaca ggcatcaggt ccccacctcc ttccctcttt gcctactcgc
      181 agaccaagga gttcatcttc tcggagctgc tggccaacct gtactcgtgt ggggaccaga
      241 acacgctgat ggaggagtcg gcggagcagg cacagcggcg cgacgagatg ctgcgcatgt
      301 accacgcact gaaggaggcg ctcagcatca tcggcgacat caacacgacc accgtcagca
      361 cgcccatgcc cccgcccgtg gacgactcct ggctgcaggt gcagagcgta ccggccggac
      421 gcaggtacca gggccggccc ccacggcccc aaagcccccc agcccggggc ccgcgggagg
      481 aatgccggga ccgggcagtg gcgcgcccgc gtcaccgggg tggctcccac ctggagcgag
      541 gggcggagct tagagagggc ggggcttgtc gcggggcggg gcttgccgtg gagagctggc
      601 tgcaaggctg ggtggagctg gggcgttggc gccgctgggg gcggggctta agctccggcg
      661 accgccttag gggtgcggca gggaggggct tgcgtgcatg ggcgtggcca gcactgggct
      721 ggggcggggc ctctgggtgg gcggagctgc tcatctcgcc tctccttgtt cctcgctccc
      781 tgtcgccctc aggtcgccca cgtccagccc cacgccgcag cgccgagccc ccgccgtgcc
      841 cccagcccgg cccgggtcgc ggggccctgc tcctgggcct ccgcctgctg ggtccgccct
      901 ggggggggcg ccccccgtgc cctccaggcc gggggcttcc cctgaccctt tcggccctcc
      961 ccctcaggtg ccctcgcgcc ccaaccgcgc cccgcccggg gtccccagcc gatcgggtca
     1021 ggcaagtcca tcccgtcctg agagccccag gccccccttc gacctcgatc ccccagtcgc
     1081 gaccttagtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1141 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     1201 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1261 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1321 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1381 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1441 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1501 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1561 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1621 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1681 ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat
     1741 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1801 gtaaagcggc cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt
     1861 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     1921 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     1981 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2041 gtatcttagc tgaatgcggc tggagaggct gccggacggg cgtggccggg agggaaatgg
     2101 ggctggattc cagagcatcg gacctggccg ccagaaccgg ccgtttctat cccaggcaat
     2161 cggactctgg gtgccggagc cacgccacgt gtggccgagg gctggcggag cctgcccccg
     2221 agggagcgct gagtcccgga ggtggtcgtt tctggggagg gggctgtggg gctcgtccca
     2281 cctgccccct tctttccagc acttgcatgg cgcttccctc tattttcact cttggcggcc
     2341 gcccacacgt tgcattcctc ctcctttctt cttcttgctg tcctccatcc tccattccgt
     2401 ccaattcctc tcccagcccc tggggagcct accttaggtc tgactctgaa ccccgatctg
     2461 ctctgagtgt gtggatttcc ttcagctacc ctgatgtccc cacttcccag ccctgactcc
     2521 tttgagccat cccagggggt gtccagccac tggcccacag gagcagaggc caggctgtga
     2581 ctgtgtgact agaaaggtgt gtgatgtgtg tgtgggcgtg cacacgagtg tgagagtgta
     2641 agcatggcac ccaggcccgg gctaggacag gagcagctgg ggaagcagcc tgggccgtgg
     2701 gcaaactgat ggcttttttc ctccccccaa ccaccccctc cgcgacttct ggccctaccc
     2761 agtctaaacc aatgtggtag gggtggcggc aggggcaggg atgggagtgc ttgaagcctg
     2821 cttcattccc aaagatttct agggaaaagc tttctactac atcctttggc ttgaccttct
     2881 tgacccatga ctctctttca aggaagttca ctccaattcc cagtctgccc tgtttccact
     2941 ggccacattc tctaaggaag aacacagcag gcggccgcca ccgcggtgga gctccagctt
     3001 ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc
     3061 tgtgtgaaat tgttatccgc tcacaattcc acacaacata ggagccggaa gcataaagtg
     3121 taaagcctgg ggtgcctaat gagtgaggta actcacatta attgcgttgc gctcactgcc
     3181 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3241 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3301 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3361 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3421 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3481 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3541 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3601 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3661 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     3721 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     3781 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     3841 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     3901 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3961 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4021 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4081 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4141 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4201 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4261 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4321 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4381 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4441 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4501 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4561 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     4621 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     4681 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     4741 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     4801 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     4861 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     4921 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4981 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5041 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5101 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5161 aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt
     5221 ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca
     5281 tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt
     5341 catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag
     5401 ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta
     5461 tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc
     5521 ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc
     5581 actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa
     5641 aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt
     5701 tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga
     5761 acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct
     5821 attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca
     5881 ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat
     5941 aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt
     6001 tatataggga tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg
     6061 aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga
     6121 aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta
     6181 gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa
     6241 atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt
     6301 gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta
     6361 cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc
     6421 cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct
     6481 gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt gatattggat
     6541 catattaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     6601 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     6661 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     6721 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     6781 tgagagtgca ccataaacga cattactata tatataatat aggaagcatt taatagacag
     6841 catcgtaata tatgtgtact ttgcagttat gacgccagat ggcagtagtg gaagatattc
     6901 tttattgaaa aatagcttgt caccttacgt acaatcttga tccggagctt ttcttttttt
     6961 gccgattaag aattaattcg gtcgaaaaaa gaaaaggaga gggccaagag ggagggcatt
     7021 ggtgactatt gagcacgtga gtatacgtga ttaagcacac aaaggcagct tggagtatgt
     7081 ctgttattaa tttcacaggt agttctggtc cattggtgaa agtttgcggc ttgcagagca
     7141 cagaggccgc agaatgtgct ctagattccg atgctgactt gctgggtatt atatgtgtgc
     7201 ccaatagaaa gagaacaatt gacccggtta ttgcaaggaa aatttcaagt cttgtaaaag
     7261 catataaaaa tagttcaggc actccgaaat acttggttgg cgtgtttcgt aatcaaccta
     7321 aggaggatgt tttggctctg gtcaatgatt acggcattga tatcgtccaa ctgcatggag
     7381 atgagtcgtg gcaagaatac caagagttcc tcggtttgcc agttattaaa agactcgtat
     7441 ttccaaaaga ctgcaacata ctactcagtg cagcttcaca gaaacctcat tcgtttattc
     7501 ccttgtttga ttcagaagca ggtgggacag gtgaactttt ggattggaac tcgatttctg
     7561 actgggttgg aaggcaagag agccccgaaa gcttacattt tatgttagct ggtggactga
     7621 cgccagaaaa tgttggtgat gcgcttagat taaatggcgt tattggtgtt gatgtaagcg
     7681 gaggtgtgga gacaaatggt gtaaaagact ctaacaaaat agcaaatttc gtcaaaaatg
     7741 ctaagaaata ggttattact gagtagtatt tatttaagta ttgtttgtgc acttgcctat
     7801 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaaac
     7861 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     7921 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     7981 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     8041 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     8101 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     8161 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     8221 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     8281 cttaatgcgc cgctacaggg cgcgtcgcgc cattcgccat tcaggctgcg caactgttgg
     8341 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     8401 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgtt
//
