LOCUS       sequence-275958-        9829 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a regulatable secretory cargo (without an ER export 
            signal) for Tetrahymena thermophila.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9829)
  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 9829)
  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-ESCargo*
FEATURES             Location/Qualifiers
     source          1..9829
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(6014..6033)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6015..6033)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6040..6057)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6040..6056)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6049..6071)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6197..6577
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6284..6303)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             6944..7738
                     /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(6998..7017)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7608..7627
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(7756..7824,7825..8616)
                     /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(7974..7993)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8787..9375
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9276..9295
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9529..9546
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    9663..9684
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(9699..9704,9705..9722,9723..9729)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9737..9753
                     /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     9742..9764
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9761..9777
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9761..9777
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        9795..9813
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     9795..9812
                     /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 aaccaatgac agcactgaaa
     2701 acgtaatcaa accattcatg agatttaaag tccacatgga aggaagtgta aatggacatg
     2761 agtttgagat agagggtgag ggagaaggaa aaccctatga gggtacccaa acagccaagc
     2821 tccaagttac caaaggtgga ccattgccct tcgcctggga catcttgagc ccacaattcc
     2881 aatatggtag taaggtctat gtaaagcacc ctgccgatat tcccgattat aagaagttgt
     2941 cttttcctga aggttttaag tgggaacgtg ttatgaattt tgaggacggt ggtgtcgtaa
     3001 ccgtaacaca agacagcagc ctccaagatg gaacctttat ctatcacgtt aagttcatcg
     3061 gtgtcaattt tccatctgac ggaccagtta tgcagaagaa gaccttgggt tgggagcctt
     3121 caaccgagcg tctctatcca agagacggag tattgaaggg tgaaattcat aaagcattga
     3181 aattaaaagg tggaggtcat tacttagtcg agtttaagtc aatatatatg gcaaaaaagc
     3241 ccgttaagtt accaggatac tactatgtcg actctaagtt agacataaca agtcacaacg
     3301 aagactatac tgtcgtagag caatacgagc gtgcagaagc ccgtcatcat ttattccaag
     3361 gttcaagtag aggtgtacaa gtcgagacca tcagtccagg agatggtcgt acctttccca
     3421 agagaggaca aaccgtcgtt gtccattaca ctggtatgtt ggaagacgga aaaaagcttg
     3481 atagcagccg tgatagaaat aaaccattca aatttatgtt gggtaaacag gaagtaattc
     3541 gtggatggga ggagggtgta gcccaaatgt cagtaggtca gcgtgctaaa ctcactataa
     3601 gcccagatta tgcttacggt gcaactggtc atccaggagt aatcccacct cacgctacct
     3661 tggtatttga cgtagagtta cttaagcttg agtgagggcc caacaaacaa ccaaccaaaa
     3721 cttctattca taaattatac taaactatat gctcatatat catacaaaat atcatatttt
     3781 ttaaatgcca tatatatgtg cgtgtacttt atttaaattt ttaactcaaa actatttttc
     3841 atataaactt attcattctt ccctataact taacaacaac gaaaaaaaaa aaatacttct
     3901 caaatattya aaamaaakat wtttamwyyy ttcataakta attttrarca ataaattctt
     3961 aaatatttca aaagcttcta gagatcttcc atacctacca gttctgcgcc tgcagcaatg
     4021 gcaacaacgt tgcccggatc cgcggccgcg gaattcctat ttaagtcagt agaattttag
     4081 atattttcat agatttttta ataagtagac aatttatttc taaaaaatat ttaaaaataa
     4141 aaaataataa gggttttgaa taactccttt aatttaaata cacattttta aatttttttt
     4201 agctctttaa atattcataa aaataaaaaa taactaacta aaaataaata aaaagataat
     4261 aatgattaaa ggtataatac tgaataagaa aaaacataat agagtactta ttttttatat
     4321 cactattttt aatatcttga aagcaaaact tttttatata tcttaaaata tattgtatcg
     4381 tttattcaat tattttcttt aaatttcaaa tatattgata aaaaagatga catgtttttt
     4441 aaagaaaaca tgaaatataa aatagataaa tatcaattat tttatttatt aaatatataa
     4501 gctgctcaaa acatagctca ttcatcaatt ataatatgtg aatcattaat tttcaaaata
     4561 ttactcatta tttaggctat catttatttt ttattttcaa ttatccgttt ctattatatt
     4621 ttaatattaa gttgtgattc ttgaattttg tgtcatgaat tatttgtaaa tctttttatt
     4681 tctggtaaaa aatataaatt gattgactca tgatttaaat catgagtcaa cctaactaat
     4741 tttcaaaatt cttctattct aaaatataga tgtgattctt gaatctctct tgaatataaa
     4801 gtaatttttt atatttctga tataattctt agctacgtga ttcacgattt atgcaatgat
     4861 ccatataaaa taatgtaaat agtgtatata tatatattcg tcttttttat tctttatata
     4921 atttaaaaaa attaaaaaaa tttaataaag ctctaataaa ataaataata atactaaact
     4981 taaaatacaa gcggccgcaa ataattttaa aacaataaat agaaaaacaa ataagattat
     5041 aaaaacttac aaaaatgcat acatttaaca tttctcaaca agatctagaa ttagtagaag
     5101 tagcgacaga gaagattaca atgctttatg aggataataa acatcatgtg ggagcggcaa
     5161 ttcgtacgaa aacaggagaa atcatttcgg cagtacatat tgaagcgtat ataggacgag
     5221 taactgtttg tgcagaagcc attgcgattg gtggtgcagt ttcgaatgga caaaaggatt
     5281 ttgacacgat tgtagctgtt agacaccctt attctgacga agtagataga agtattcgag
     5341 tggtaagtcc ttgtggtatg tgtagggagt tgatttcaga ctatgcacca gattgttttg
     5401 tgttaataga aatgaatggc aagttagtca aaactacgat tgaagaactc attccactca
     5461 aatatacccg aaattgactc gagtgattga ttgagataaa gctaaggctt gctgtactga
     5521 tccaaacagt ggatgttgct gctcaaataa ggcaaataaa tgctgtgatg ctacttcaaa
     5581 gcaagagtgt tagacttgtt aatgttgtaa atgaaataaa tatgttaatt aaaatttaaa
     5641 atatgttgat gttgtaattc ataattgcat aaaatcagtt tcgtatttta ttaaaaactt
     5701 taatattatc atcctaaaat ttattaaaaa aaactgttat taataaatca ataattatcg
     5761 ctatttcata gattttttta aatgcaaaag caagtatgtt atttaaatta agtaaacatt
     5821 gattgaaagt attatctaca gaaaaaaaaa tatataattt actttttaat caatttaatt
     5881 ggaaattatt ttatattttt taaaaatgaa aaaacatata tatttataat aaacattcaa
     5941 atttatttta caaaatttat ataaaaatct ttaataatta caattaatta ggccgccgtg
     6001 gccgcccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg
     6061 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     6121 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     6181 cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     6241 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     6301 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     6361 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     6421 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     6481 acgttcttta atagtggatt cttgttccaa actggaacaa cactcaaccc tatctcggtc
     6541 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     6601 atttaacaaa aaywkrrcgc rarkkytrmy aaarkwmrcg gaacacgtag aaagccagtc
     6661 cgcagaaacg gtgctgaccc cgatgaatgt cagctactgg gctatctgga caagggaaaa
     6721 cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat agctagactg
     6781 ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt
     6841 tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag
     6901 gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     6961 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     7021 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     7081 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg
     7141 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     7201 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatccca
     7261 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     7321 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     7381 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     7441 gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt
     7501 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     7561 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     7621 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     7681 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgaat
     7741 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     7801 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     7861 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     7921 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     7981 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     8041 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     8101 actgtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     8161 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     8221 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     8281 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     8341 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     8401 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     8461 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     8521 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     8581 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     8641 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     8701 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     8761 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     8821 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     8881 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     8941 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     9001 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     9061 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     9121 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     9181 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     9241 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     9301 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     9361 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     9421 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     9481 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     9541 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     9601 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     9661 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     9721 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     9781 gattacgcca agctatttag gtgacactat agaatactca agctatgca
//
