LOCUS       sequence-355814-        6655 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Synchronize trafficking of TNFa-RpHLuorin2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6655)
  AUTHORS   Linders PTA, Ioannidis M, Ter Beest M, van den Bogaart G
  TITLE     Fluorescence Lifetime Imaging of pH along the Secretory Pathway.
  JOURNAL   ACS Chem Biol. 2022 Jan 21;17(1):240-251. doi: 
            10.1021/acschembio.1c00907. Epub 2022 Jan 10.
  PUBMED    35000377
REFERENCE   2  (bases 1 to 6655)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acschembio.1c00907"; journalName: "ACS Chem Biol"; date: 
            "2022-01-21- 21"; volume: "17"; issue: "1"; pages: "240-251"
COMMENT     Sequence Label: Str-KDEL_TNF-SBP-RpHLuorin2
FEATURES             Location/Qualifiers
     source          1..6655
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(127..146)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     852..872
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     946..965
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        946..964
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     intron          1729..1958
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     1941..1960
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2030..2604,2605..2607,2608..2616)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2197..2214)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2424..2443
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3374..3484
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="DEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     primer_bind     complement(3794..3813)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    4358..4479
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4395..4414)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4581..4597)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4581..4597)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4594..4616)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4605..4621
                     /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(4629..4635,4636..4653,4654..4659))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4674..4695
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4812..4829)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4983..5571)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5063..5082)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5742..6533,6534..6602))
                     /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     6365..6384
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6603..6655,1..52))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
       61 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
      121 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
      181 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
      241 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
      301 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
      361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      541 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      841 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      901 tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata cgactcacta
      961 tagggagacc caagcttggt accgagctcg gatcgatatc tgcggcctag ctagcgctta
     1021 aggcctgtta accggtatgg atgtatgcgt ccgtcttgcc ctgtggctcc tctggggact
     1081 cctcctgcac cagggccaga gcctcagcca tagtcacagt gagaaggcga caggaaccag
     1141 ctcgggcgcc ggtttaaacg accctagcaa agactcaaaa gctcaggtgt ccgctgccga
     1201 ggctggcatt actggaacat ggtacaatca gctcgggagc acctttattg tgactgctgg
     1261 agccgatgga gccctcaccg gaacatacga atctgctgtg ggaaacgccg aatcacggta
     1321 cgtcctcact ggccgatacg atagtgcccc tgccaccgac ggatctggga ctgccctggg
     1381 atggactgtc gcttggaaaa acaactaccg gaatgctcat tctgccacaa catggagtgg
     1441 acagtacgtg ggaggcgctg aggctagaat caatacacag tggctgctca catctggcac
     1501 aaccgaggca aatgcttgga aatccaccct ggtgggacat gacacattca ccaaagtgaa
     1561 accctccgcc gcttcaatcg atgccgccaa aaaagccgga gtcaacaacg gcaatcctct
     1621 ggatgccgtc cagcagctcg gaccgaaaga tgaactgtga gcggccgcat agataactga
     1681 tccagtgtgc tggaattaat tcgctgtctg cgagggccag ctgttggggt gagtactccc
     1741 tctcaaaagc gggcatgact tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt
     1801 tgatattcac ctggcccgcg gtgatgcctt tgagggtggc cgcgtccatc tggtcagaaa
     1861 agacaatctt tttgttgtca agcttgaggt gtggcaggct tgagatctgg ccatacactt
     1921 gagtgacaat gacatccact ttgcctttct ctccacaggt gtccactccc aggtccaact
     1981 gcaggtcgag catgcatcta gggcggccaa ttccgcccct ctcccccccc ccctctccct
     2041 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     2101 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     2161 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     2221 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
     2281 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     2341 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     2401 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     2461 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta
     2521 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     2581 tcctttgaaa aacacgatga taagcttgcc acaacccggg aggcgcgcca tgtctaccga
     2641 gtctatgatt agggacgtgg aactggctga ggaggcactg cccaaaaaaa ccggcggacc
     2701 acagggctct aggagatgtc tgtttctgtc tctgttctct tttctcatcg tggctggcgc
     2761 tacaacactc ttctgtctgc tccatttcgg cgtgattgga ccacagcgag aggaatttcc
     2821 ccgggatctg tcactcattt caccactggc acaggctgtc cgatcttcat ctcggactcc
     2881 atccgacaaa cctgtcgccc atgtcgtcgc caacccacag gccgagggcc agctccagtg
     2941 gctcaatagg agggcaaacg ctctgctcgc caatggcgtg gaactccggg ataaccagct
     3001 cgtcgtgcct agtgagggac tgtacctcat ctactcccag gtgctgttta agggccaggg
     3061 atgtccttct acacatgtgc tgctcacaca cacaatttca cggatcgccg tgtcttacca
     3121 gactaaagtc aatctgctct ctgccatcaa atccccatgt cagcgggaaa cacctgaggg
     3181 cgctgaggct aaaccttggt acgaacccat ctacctcgga ggcgtgttcc agctggagaa
     3241 gggcgataga ctgagtgctg agatcaatcg acccgactac ctcgactttg ccgaatctgg
     3301 ccaggtctac tttggcatca ttgctctggg cctcgagggc cgagctcatg gcgcacctag
     3361 gcctttgaat tccgacgaga agaccactgg ttggcgaggt ggacacgttg ttgaaggact
     3421 ggctggggaa cttgaacaac ttcgtgcacg actggagcat cacccacaag gtcaacgtga
     3481 accacctgca ggtagcaagg gcgaggagct gttcaccggc gtggtgccca tcctggtgga
     3541 gctggacggc gacgtgaacg gccacaagtt cagcgtgagc ggcgagggcg agggcgacgc
     3601 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     3661 gcccaccctg gtgaccaccc tgagctacgg cgtgcagtgc ttcagccgct accccgacca
     3721 catgaagcag cacgacttct tcaagagcgc catgcccgag ggctacgtgc aggagcgcac
     3781 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     3841 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     3901 gggccacaag ctggagtaca actacaacga gcacctggtc tacatcatgg ccgacaagca
     3961 gaagaacggc accaaggcca tcttccaggt gcaccacaac atcgaggacg gcggcgtgca
     4021 gctggccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     4081 caaccactac ctgcacaccc agagcgccct gagcaaggac cccaacgaga agcgcgacca
     4141 catggtgctg ctggagttcg tgaccgccgc cggcatcacc cacggcatgg acgagctgta
     4201 caagggccgg ccttaaggcc tcgagggcca attaattaac tctagataac tgatcataat
     4261 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     4321 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     4381 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4441 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgcgtcgagt gcattctagt
     4501 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4561 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4621 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4681 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4741 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4801 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4861 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4921 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4981 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5041 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5101 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5161 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5221 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5281 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5341 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5401 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5461 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5521 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5581 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5641 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5701 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5761 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5821 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5881 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5941 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6001 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     6061 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6121 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6181 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6241 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6301 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6361 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6421 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6481 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6541 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6601 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catga
//
