LOCUS       sequence-275964-        9847 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a regulatable secretory cargo for Tetrahymena thermophila 
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9847)
  AUTHORS   Casler JC, Zajac AL, Valbuena FM, Sparvoli D, Jeyifous O, Turkewitz 
            AP, Horne-Badovinac S, Green WN, Glick BS
  TITLE     ESCargo: a regulatable fluorescent secretory cargo for diverse model
            organisms.
  JOURNAL   Mol Biol Cell. 2020 Oct 28:mbcE20090591. doi: 
            10.1091/mbc.E20-09-0591.
  PUBMED    33112725
REFERENCE   2  (bases 1 to 9847)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Biol 
            Cell. 2020 Oct 28:mbcE20090591. doi: 10.1091/mbc.E20-09-0591."
COMMENT     Sequence Label: ncvB-ssGrl1-APVNTT-DsRed-Express2-FKBP(LV)(C22V)
FEATURES             Location/Qualifiers
     source          1..9847
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(6032..6051)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6033..6051)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6058..6075)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6058..6074)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6067..6089)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6215..6595
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6302..6321)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             6962..7756
                     /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"
     primer_bind     complement(7016..7035)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7626..7645
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(7774..7842,7843..8634)
                     /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(7992..8011)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8805..9393
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9294..9313
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9547..9564
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9681..9702
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9717..9722,9723..9740,9741..9747)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9755..9771
                     /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     9760..9782
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9779..9795
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9779..9795
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        9813..9831
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     9813..9830
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 ggccgttttg gcctttttta tttagtaaaa atttatcttt catttaattt ataatcatat
       61 ttatatcttt gtatttaaaa acaataaata atagcaatgg ctatactatc cttgactccc
      121 attaatcatt tgtgataata aaataagaaa atggaaataa ttgagatata aaatcatata
      181 gcaacgctct tgattctttg tagtgtttaa aaatagtttg cctacttatg catagtatac
      241 ctttgacata tattcctgta tttgcatgaa caaacatatc taaaaacaaa attaactaac
      301 aaataagaat aatatctaat ctaaatagtt aatgaagctg agcaccaaag ctaaagcaaa
      361 caaaatagca aaaaaagcat ataagagatg caaaaatttt taaagcctac caaacttcta
      421 tgcatcttgt atgtggtaaa aaaataggta tttatttata gaaatattct aaacaattaa
      481 ttttctactt gctcttttta actgctttgt caaaataaat catagtatta tacaatttac
      541 ttttttcaga tattattttt taactgaaca ttttattagc ttaaaaatat tgatgctttt
      601 catccttaaa atgagatctg tcatttatca tttctgttta cctttatgtt attagtcttt
      661 gcattctact tcgagcaaac tataaaaata tttgagcata ctttctaaat ctaattttaa
      721 aagatatttc ttaaagtctt taaagttagt ttttttattg aagaataatt agctttaatc
      781 aagtaaaatg ttttattgtt aatttattta attagattta ttttataagt tattttggct
      841 atggttagca aattatatct ttaaatcctt tttcatactt attttgtata ttcaaaatct
      901 caacaaaagc taatttctac tttttaatta attaatttct actaatttag taaaaaataa
      961 aaatttgcaa attttttaat taattaaaaa tcctttgtaa cgttttgatg taattttaag
     1021 agtccttggt cttaacacta aaaacttatt taaagaaaaa taaatcaata atgcaaattt
     1081 attttattca aggcttgctt ttaaagcatt aaataggatt aaacaatata aattttagct
     1141 atttaaatta aatttattat ttttaatgat taaaattatt aaacttgaat aaatgatcct
     1201 aattcttttt gttatttata ttggaataac atttaagtaa atcaacataa aaatgacttt
     1261 aatacaattt actattcttc atagtagttg cttgagcttc tgaagtatga attaggttta
     1321 aatatcatat tattttagct gtttaaatta aatttattat ttttgatgat taaaataatt
     1381 cagattttat gaatgatact acttatttta aatttcatgt aaaaataata ttagtgatgg
     1441 tattaaaaat ataattagat tttttaaaaa ttgtttggaa ataagtactt aattccaaat
     1501 ttcttttaat ttttaagaat taaattagtt tatttttata tattaattca ctaaataaaa
     1561 agataagtaa tatatttagt gcacaatgtt tgaatgtttt tttgataaat ttgtaaatta
     1621 aatctattta agtcagtaga attttagata ttttcataga ttttttaata agtagacaat
     1681 ttatttctaa aaaatattta aaaataaaaa ataataaggg ttttgaataa ctcctttaat
     1741 ttaaatacac atttttaaat tttttttagc tctttaaata ttcataaaaa taaaaaataa
     1801 ctaactaaaa ataaataaaa agataataat ggttaaaggt ataatactga ataagaaaaa
     1861 acataataga gtacttattt tttatatcac tatttttaat atcttgaaag caaaactttt
     1921 ttatatatct taaaatatat tgtatcgttt attcaattat tttctttaaa tttcaaatat
     1981 attgataaaa aagatgacat gttttttaaa gaaaacatga aatataaaat agataaatat
     2041 caattatttt atttattaaa tatataagct gctcaaaaca tagctcattc atcaattata
     2101 atatgtgaat cattaatttt caaaatatta ctcattattt aggctatcat ttatttttta
     2161 ttttcaatta tccgtttcta ttatatttta atattaagtt gtgattcttg aattttgtgt
     2221 catgaattat ttgtaaatct ttttatttct gataaaaaat ataaattgat tgactcatga
     2281 tttaaatcat gagtcaacct aactaatttt caaaattctt ctattctaaa atatagatgt
     2341 gattcttgaa tctctcttga atataaagta attttttata tttctgatat aattcttagc
     2401 tacgtgattc acgatttatg caatgatcca tataaaataa tgtaaatagt gtatatatat
     2461 atattcgtcc tttttattct ttatataatt taaaaaaatt aaaaaaattt aataaagctc
     2521 taataaaata aataataata ctaaacttaa aatacaagcg caaataattt taaaacaata
     2581 aatagaaaaa caaataagat tataaaaact tacaaaagtt taaactgatg aataaaaagc
     2641 tcttagtcgt cttatttggt tttttggcat tagccgcagc aaccaatgct ccagtaaata
     2701 caactgacag cactgaaaac gtaatcaaac cattcatgag atttaaagtc cacatggaag
     2761 gaagtgtaaa tggacatgag tttgagatag agggtgaggg agaaggaaaa ccctatgagg
     2821 gtacccaaac agccaagctc caagttacca aaggtggacc attgcccttc gcctgggaca
     2881 tcttgagccc acaattccaa tatggtagta aggtctatgt aaagcaccct gccgatattc
     2941 ccgattataa gaagttgtct tttcctgaag gttttaagtg ggaacgtgtt atgaattttg
     3001 aggacggtgg tgtcgtaacc gtaacacaag acagcagcct ccaagatgga acctttatct
     3061 atcacgttaa gttcatcggt gtcaattttc catctgacgg accagttatg cagaagaaga
     3121 ccttgggttg ggagccttca accgagcgtc tctatccaag agacggagta ttgaagggtg
     3181 aaattcataa agcattgaaa ttaaaaggtg gaggtcatta cttagtcgag tttaagtcaa
     3241 tatatatggc aaaaaagccc gttaagttac caggatacta ctatgtcgac tctaagttag
     3301 acataacaag tcacaacgaa gactatactg tcgtagagca atacgagcgt gcagaagccc
     3361 gtcatcattt attccaaggt tcaagtagag gtgtacaagt cgagaccatc agtccaggag
     3421 atggtcgtac ctttcccaag agaggacaaa ccgtcgttgt ccattacact ggtatgttgg
     3481 aagacggaaa aaagcttgat agcagccgtg atagaaataa accattcaaa tttatgttgg
     3541 gtaaacagga agtaattcgt ggatgggagg agggtgtagc ccaaatgtca gtaggtcagc
     3601 gtgctaaact cactataagc ccagattatg cttacggtgc aactggtcat ccaggagtaa
     3661 tcccacctca cgctaccttg gtatttgacg tagagttact taagcttgag tgagggccca
     3721 acaaacaacc aaccaaaact tctattcata aattatacta aactatatgc tcatatatca
     3781 tacaaaatat catatttttt aaatgccata tatatgtgcg tgtactttat ttaaattttt
     3841 aactcaaaac tatttttcat ataaacttat tcattcttcc ctataactta acaacaacga
     3901 aaaaaaaaaa atamttctca aatwtwyaaa wmrwakwtwt ttaatccctt cataagtaat
     3961 tttgagcaat aaattcttaa atatttcaaa agcttctaga gatcttccat acctaccagt
     4021 tctgcgcctg cagcaatggc aacaacgttg cccggatccg cggccgcgga attcctattt
     4081 aagtcagtag aattttagat attttcatag attttttaat aagtagacaa tttatttcta
     4141 aaaaatattt aaaaataaaa aataataagg gttttgaata actcctttaa tttaaataca
     4201 catttttaaa ttttttttag ctctttaaat attcataaaa ataaaaaata actaactaaa
     4261 aataaataaa aagataataa tgattaaagg tataatactg aataagaaaa aacataatag
     4321 agtacttatt ttttatatca ctatttttaa tatcttgaaa gcaaaacttt tttatatatc
     4381 ttaaaatata ttgtatcgtt tattcaatta ttttctttaa atttcaaata tattgataaa
     4441 aaagatgaca tgttttttaa agaaaacatg aaatataaaa tagataaata tcaattattt
     4501 tatttattaa atatataagc tgctcaaaac atagctcatt catcaattat aatatgtgaa
     4561 tcattaattt tcaaaatatt actcattatt taggctatca tttatttttt attttcaatt
     4621 atccgtttct attatatttt aatattaagt tgtgattctt gaattttgtg tcatgaatta
     4681 tttgtaaatc tttttatttc tggtaaaaaa tataaattga ttgactcatg atttaaatca
     4741 tgagtcaacc taactaattt tcaaaattct tctattctaa aatatagatg tgattcttga
     4801 atctctcttg aatataaagt aattttttat atttctgata taattcttag ctacgtgatt
     4861 cacgatttat gcaatgatcc atataaaata atgtaaatag tgtatatata tatattcgtc
     4921 ttttttattc tttatataat ttaaaaaaat taaaaaaatt taataaagct ctaataaaat
     4981 aaataataat actaaactta aaatacaagc ggccgcaaat aattttaaaa caataaatag
     5041 aaaaacaaat aagattataa aaacttacaa aaatgcatac atttaacatt tctcaacaag
     5101 atctagaatt agtagaagta gcgacagaga agattacaat gctttatgag gataataaac
     5161 atcatgtggg agcggcaatt cgtacgaaaa caggagaaat catttcggca gtacatattg
     5221 aagcgtatat aggacgagta actgtttgtg cagaagccat tgcgattggt ggtgcagttt
     5281 cgaatggaca aaaggatttt gacacgattg tagctgttag acacccttat tctgacgaag
     5341 tagatagaag tattcgagtg gtaagtcctt gtggtatgtg tagggagttg atttcagact
     5401 atgcaccaga ttgttttgtg ttaatagaaa tgaatggcaa gttagtcaaa actacgattg
     5461 aagaactcat tccactcaaa tatacccgaa attgactcga gtgattgatt gagataaagc
     5521 taaggcttgc tgtactgatc caaacagtgg atgttgctgc tcaaataagg caaataaatg
     5581 ctgtgatgct acttcaaagc aagagtgtta gacttgttaa tgttgtaaat gaaataaata
     5641 tgttaattaa aatttaaaat atgttgatgt tgtaattcat aattgcataa aatcagtttc
     5701 gtattttatt aaaaacttta atattatcat cctaaaattt attaaaaaaa actgttatta
     5761 ataaatcaat aattatcgct atttcataga tttttttaaa tgcaaaagca agtatgttat
     5821 ttaaattaag taaacattga ttgaaagtat tatctacaga aaaaaaaata tataatttac
     5881 tttttaatca atttaattgg aaattatttt atatttttta aaaatgaaaa aacatatata
     5941 tttataataa acattcaaat ttattttaca aaatttatat aaaaatcttt aataattaca
     6001 attaattagg ccgccgtggc cgcccaattc gccctatagt gagtcgtatt acaattcact
     6061 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     6121 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     6181 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc
     6241 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     6301 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     6361 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     6421 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     6481 cctttgacgt tggagtccac gttctttaat agtggattct tgttccaaac tggaacaaca
     6541 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     6601 tggttaaaaa atgagctgat ttaacaaaww ywkrrcgcra rkkytrmyaa aggaagcgga
     6661 acacgtagaa agccagtccg cagaaacggt gctgaccccg atgaatgtca gctactgggc
     6721 tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac
     6781 atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg
     6841 ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc
     6901 aaggatctga tggcgcaggg gatcaagatc tgatcaagag acaggatgag gatcgtttcg
     6961 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     7021 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     7081 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     7141 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     7201 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     7261 ggatctcctg tcatcccacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     7321 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     7381 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     7441 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     7501 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     7561 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     7621 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     7681 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     7741 tgacgagttc ttctgaattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     7801 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     7861 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     7921 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     7981 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     8041 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     8101 catcttacgg atggcatgac tgtaagagaa ttatgcagtg ctgccataac catgagtgat
     8161 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     8221 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     8281 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     8341 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     8401 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     8461 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     8521 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     8581 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     8641 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     8701 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     8761 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     8821 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     8881 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     8941 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     9001 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     9061 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     9121 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     9181 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     9241 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     9301 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     9361 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     9421 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     9481 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     9541 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     9601 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     9661 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     9721 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     9781 ggaaacagct atgaccatga ttacgccaag ctatttaggt gacactatag aatactcaag
     9841 ctatgca
//
