LOCUS       pcDNAmTFP1Rab5c         6755 bp    DNA     circular SYN 05-JUL-2024
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6755)
  AUTHORS   Alessandra Affolter Lab
  TITLE     Direct Submission
  JOURNAL   Exported Jun 11, 2025 from SnapGene 8.1.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6755
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     primer_bind     135..152
                     /label=pcdnafor
     primer_bind     223..263
                     /label=testcmvpromfor
     enhancer        join(235..249,250..614)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    join(236..249,250..852)
                     /label=cmv_immearly_prom
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     726..745
                     /label=EGFP-N-for
     primer_bind     769..789
                     /label=CMV-for
     primer_bind     769..789
                     /label=CMVfor
     primer_bind     863..882
                     /label=T7
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     885..922
                     /label=testtfprab5cmvfor
     primer_bind     complement(886..922)
                     /label=testcmvpromrev
     primer_bind     900..919
                     /label=mcherry5pletm1_for
     CDS             902..1607
                     /label=mTFP1
     misc_feature    902..1602
                     /label=mTFP
     primer_bind     902..935
                     /label=mTFPrab5for
     misc_feature    902..922
                     /label=GFP
     CDS             join(902..904,905..907,908..922)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEE"
     primer_bind     902..922
                     /label=mTFPmito_for
     primer_bind     1288..1297
                     /label=mcherry5pletm1_for
     primer_bind     complement(1581..1608)
                     /label=mtfprab5f.REVa
     primer_bind     complement(1581..1608)
                     /label=mtfprab5f.REV
     primer_bind     complement(1588..1632)
                     /label=testtfprabcmvrev
     primer_bind     complement(1591..1608)
                     /label=mTFPmito_rev
     primer_bind     complement(1595..1630)
                     /label=mcherry3pletm1_reva
     primer_bind     complement(1595..1608)
                     /label=mcherry3pletm1_rev
     primer_bind     1602..1629
                     /label=fRAB5Cfor
     misc_feature    1603..1611
                     /label=attB2/P2/L2 shared
                     /note="attB2/P2/L2 shared"
     CDS             1603..1606
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="Y"
     misc_feature    1619..2284
                     /label=Rab5c
                     /note="Rab5c"
     primer_bind     complement(1631..1651)
                     /label=seqrab5rev
     primer_bind     complement(2268..2291)
                     /label=fRAB5Crev
     promoter        complement(2307..2325)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2308..2325)
                     /label=SP6
     primer_bind     complement(2345..2362)
                     /label=BGH-rev
     polyA_signal    2351..2575
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2621..3049
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     rep_origin      2638..2944
                     /label=F1 ori
     promoter        3063..3393
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3244..3379
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3460..4254
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3463..4254
                     /label=Kan/neoR
     polyA_signal    4428..4549
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4428..4445)
                     /label=EGFP-C-Rev
     primer_bind     complement(4594..4614)
                     /label=M13-rev
     primer_bind     complement(4598..4614)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_binding    complement(4620..4642)
                     /label=LacO
     protein_bind    4622..4638
                     /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(join(4646..4652,4653..4670,4671..4676))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4691..4712
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4982..5664)
                     /direction=LEFT
                     /label=ColE1 origin
     rep_origin      complement(5000..5588)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5759..6550,6551..6619))
                     /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"
     CDS             complement(5762..6421)
                     /label=AmpR
     promoter        complement(6620..6724)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 catggtgagc aagggcgagg agaccacaat gggcgtaatc aagcccgaca tgaagatcaa
      961 gctgaagatg gagggcaacg tgaatggcca cgccttcgtg atcgagggcg agggcgaggg
     1021 caagccctac gacggcacca acaccatcaa cctggaggtg aaggagggag cccccctgcc
     1081 cttctcctac gacattctga ccaccgcgtt cgcctacggc aacagggcct tcaccaagta
     1141 ccccgacgac atccccaact acttcaagca gtccttcccc gagggctact cttgggagcg
     1201 caccatgacc ttcgaggaca agggcatcgt gaaggtgaag tccgacatct ccatggagga
     1261 ggactccttc atctacgaga tacacctcaa gggcgagaac ttccccccca acggccccgt
     1321 gatgcagaag aagaccaccg gctgggacgc ctccaccgag aggatgtacg tgcgcgacgg
     1381 cgtgctgaag ggcgacgtca agcacaagct gctgctggag ggcggcggcc accaccgcgt
     1441 tgacttcaag accatctaca gggccaagaa ggcggtgaag ctgcccgact atcactttgt
     1501 ggaccaccgc atcgagatcc tgaaccacga caaggactac aacaaggtga ccgtttacga
     1561 gagcgccgtg gcccgcaact ccaccgacgg catggacgag ctgtacaaag tggtcaccat
     1621 ggcggggcga ggtggaccag cgcggaccaa cggtgccgcc cccgtcggga acaaaatctg
     1681 ccagtttaag cttgtgttgc tgggagagtc tgcagtaggc aagtccagcc tggtgctgcg
     1741 cttcgtcaaa ggccagtttc acgagtacca ggagagcacc attggagctg ctttcctcac
     1801 acagacactc tgcttggacg atacgacagt aaagtttgag atttgggaca cggccggaca
     1861 ggagcggtat cacagtttgg cccctatgta ttacagagga gcccaggcag ccatcgtcgt
     1921 ctacgacatt actaacacag acacattcac tagagcaaag aactgggtga aggagctgca
     1981 gcgccaggcc agtccaaata tcgttattgc tctggcaggc aacaaagcag atctggccaa
     2041 caagagagcc gttgactttc aggaggccca ggcatacgca gatgacaaca gcctactctt
     2101 catggagacg tctgcaaaga ccgccatgaa cgtcaatgag atcttcatgg ctatcgccaa
     2161 gaagcttccg aaaagcgagc cgcagggtgg agcgggcagc gggggacggg cccgaggagg
     2221 ggtggactta caggagacgg cgcctcaggg aagaagcggc cagtgctgtg gaggcggaaa
     2281 ctaaggaatt ctctagaggg ccctattcta tagtgtcacc taaatgctag agctcgctga
     2341 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2401 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2461 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2521 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2581 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2641 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2701 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2761 caagctctaa atcggggcat ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2821 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2881 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2941 acaacactca accctatctc ggtctattct tttgatttat aagggatttt ggggatttcg
     3001 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     3061 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc aggcaggcag aagtatgcaa
     3121 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3181 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3241 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3301 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     3361 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     3421 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     3481 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     3541 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     3601 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     3661 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     3721 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     3781 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     3841 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     3901 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3961 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     4021 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     4081 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     4141 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     4201 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     4261 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     4321 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     4381 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     4441 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4501 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     4561 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     4621 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     4681 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4741 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4801 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4861 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4921 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4981 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5041 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5101 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5161 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
     5221 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5281 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5341 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5401 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     5461 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5521 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5581 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5641 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5701 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5761 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5821 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5881 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5941 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6001 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6061 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6121 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6181 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6241 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6301 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6361 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6421 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6481 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6541 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6601 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6661 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6721 cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
