LOCUS       pET_SUMO-syt1_C2        6671 bp DNA     circular SYN 10-NOV-2021
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6671)
  AUTHORS   Chapman lab
  TITLE     Direct Submission
  JOURNAL   Exported Sep 12, 2023 from SnapGene 7.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     CDS             complement(548..1363)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /note="color: #ccffcc"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      1485..2073
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(2500..2691)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /note="color: #993366"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3500..4582)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /note="color: #993366"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(4583..4660)
                     /gene="lacI"
                     /label=lacI promoter
                     /note="color: #ffffff; direction: LEFT"
     promoter        4969..4987
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     protein_bind    4988..5012
                     /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)."
                     /note="color: #31849b"
     RBS             5042..5048
                     /note="ribosome binding site"
                     /note="color: #a6acb3"
     CDS             5057..5059
                     /codon_start=1
                     /product="start codon"
                     /label=ATG
                     /note="color: #993366"
                     /translation="M"
     CDS             5069..5086
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     primer_bind     complement(5075..5141)
                     /label=HRV3c insertion-R
                     /note="CTGGAAGTTCTGTTCCaGGGGCCCLeuGluValLeuPheGln/GlyPro."
                     /note="color: black; sequence: 
                     GATTGACTTCTGAGTCCGACATGCTAGCGGGCCCCtGGAACAGAACTTCCAGGCCGCTG
                     CCGTGATGATGATG"
     primer_bind     5075..5141
                     /label=HRV3c insertion-F
                     /note="CTGGAAGTTCTGTTCCaGGGGCCCLeuGluValLeuPheGln/GlyPro."
                     /note="color: black; sequence: 
                     CATCATCATCACGGCAGCGGCCTGGAAGTTCTGTTCCaGGGGCCCGCTAGCATGTCGGA
                     CTCAGAAGTCAATC"
     CDS             5096..5113
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /note="color: #cc99b2
                     Cleavage site after base 5107"
                     /translation="LVPRGS"
     CDS             5120..5413
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="ubiquitin-like protein tag"
                     /label=SUMO
                     /note="color: #cc99b2
                     Cleavage site after base 5413"
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     5309..5331
                     /label=SUMO Forward
                     /note="color: black; sequence: AGATTCTTGTACGACGGTATTAG"
     primer_bind     5401..5449
                     /label=Fragment.FOR
                     /note="color: black; sequence: 
                     ACAGATTGGTGGatcCAACCAATGTTTGTTCCAATCGACCAACAACACT"
     misc_feature    5413..5415
                     /label=BamHI mutation
                     /note="color: #a6acb3"
     CDS             5417..6442
                     /codon_start=1
                     /label=syt1 80-421-Juxta Lys
                     /note="color: #a6acb3"
                     /translation="NQCLFQSTNNTQGSENGGNNAINMQDVNDLGSTMTDQALNDDDAE
                     TGLTDGEENEEPTEEEKLGKLQYSLDYDFQNNQLLVGIIQAAELPALDMGGTSDPYVKV
                     FLLPDKKKKFETKVHRKTLNPVFNEQFTFKVPYSELGGKTLVMAVYDFDRFSKHDIIGE
                     FKVPMNTVDFGHVTEEWRDLQSAEKEEQEKLGDICFSLRYVPTAGKLTVVILEAKNLKK
                     MDVGGLSDPYVKIHLMQNGKRLKKKKTTIKKNTLNPYYNESFSFEVPFEQIQKVQVVVT
                     VLDYDKIGKNDAIGKVFVGYNSTGAELRHWSDMLANPRRPIAQWHTLQVEEEVDAMLAV
                     KK"
     CDS             6504..6521
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     primer_bind     complement(6571..6590)
                     /label=T7 Reverse
                     /note="color: black; sequence: TAGTTATTGCTCAGCGGTGG"
     terminator      6588..6635
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
                     /note="color: #ffffff"
ORIGIN
        1 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
       61 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
      121 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
      181 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
      241 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
      541 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
      601 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
      661 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
      721 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
      781 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
      841 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
      901 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
      961 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     1021 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     1081 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     1141 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     1201 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     1261 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     1321 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     1381 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     1441 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     1501 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     1561 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     1621 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     1681 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     1741 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     1801 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     1861 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     1921 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     1981 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     2041 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     2101 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     2161 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     2221 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     2281 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     2341 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     2401 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2461 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     2521 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     2581 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     2641 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     2701 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     2761 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     2821 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     2881 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     2941 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     3001 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     3061 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     3121 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     3181 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     3241 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg
     3301 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     3361 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     3421 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     3481 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3541 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     3601 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     3661 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     3721 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     3781 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     3841 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     3901 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     3961 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
     4021 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
     4081 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
     4141 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
     4201 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
     4261 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
     4321 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
     4381 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
     4441 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
     4501 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     4561 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     4621 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     4681 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     4741 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     4801 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     4861 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     4921 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     4981 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     5041 agaaggagat atacatatgg gcagcagcca tcatcatcat catcacggca gcggcctggt
     5101 gccgcgcggc agcgctagca tgtcggactc agaagtcaat caagaagcta agccagaggt
     5161 caagccagaa gtcaagcctg agactcacat caatttaaag gtgtccgatg gatcttcaga
     5221 gatcttcttc aagatcaaaa agaccactcc tttaagaagg ctgatggaag cgttcgctaa
     5281 aagacagggt aaggaaatgg actccttaag attcttgtac gacggtatta gaattcaagc
     5341 tgatcagacc cctgaagatt tggacatgga ggataacgat attattgagg ctcacagaga
     5401 acagattggt ggatccaacc aatgtttgtt ccaatcgacc aacaacactc aggggtcgga
     5461 aaacggagga aacaacgcca ttaacatgca agacgtgaac gacttagggt cgaccatgac
     5521 tgatcaggcc cttaatgatg acgatgctga aaccggactg actgatggag aagaaaacga
     5581 agagcccact gaagaggaga aactgggaaa gctccaatat tcactggact atgacttcca
     5641 gaataaccag ctgttggtgg gaatcatcca ggctgctgaa ctgcccgccc tggacatggg
     5701 gggtacatcc gatccatacg tcaaagtctt cctgctgcct gacaaaaaga agaaatttga
     5761 gactaaagtc caccggaaaa ccctcaatcc agtcttcaat gaacaattta ctttcaaggt
     5821 gccctactcg gaattaggtg gcaaaaccct ggtgatggct gtgtatgact ttgatcgctt
     5881 ctccaagcat gacatcatcg gagagttcaa agttcctatg aacaccgtgg attttggcca
     5941 tgtgaccgag gagtggcgtg atctccagag cgctgagaaa gaagagcaag agaaactggg
     6001 tgacatctgc ttctccctcc gctacgtccc tactgccggc aaactgactg ttgtcattct
     6061 ggaagccaag aacctgaaga agatggatgt gggtggctta tctgatccct acgtgaagat
     6121 tcacctgatg cagaacggta agaggctgaa gaagaaaaag acgacgatta agaagaacac
     6181 actcaacccc tactacaacg agtccttcag ctttgaagtt ccgttcgagc aaatccagaa
     6241 agtgcaagtg gtggtaactg ttttggacta tgacaagatt ggcaagaacg acgccatcgg
     6301 caaagtcttc gttggttaca acagcactgg ggcggagctg cgacactggt cagacatgct
     6361 ggccaacccc cggcgaccca tcgcacagtg gcacactctg caggtagagg aggaggttga
     6421 tgccatgctg gctgtcaaga agtgaaagct taggtattta ttcggcgcaa agtgcgtcgg
     6481 gtgatgctgc caacttagtc gagcaccacc accaccacca ctgagatccg gctgctaaca
     6541 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     6601 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     6661 tggcgaatgg g
//
