LOCUS       sequence-388757-        7094 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For affinity purification of Pdp1 payload.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7094)
  AUTHORS   Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, Zhang F
  TITLE     Programmable protein delivery with a bacterial contractile injection
            system.
  JOURNAL   Nature. 2023 Apr;616(7956):357-364. doi: 10.1038/s41586-023-05870-7.
            Epub 2023 Mar 29.
  PUBMED    36991127
REFERENCE   2  (bases 1 to 7094)
  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"; doi: 
            "10.1038/s41586-023-05870-7"; journalName: "Nature"; date: 
            "2023-04"; volume: "616"; issue: "7956"; pages: "357-364"
COMMENT     Sequence Label: TwinStrep-SUMO-Pdp1
FEATURES             Location/Qualifiers
     source          1..7094
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(188..235)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     231..249
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1310..1603)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(join(1604..1627,1628..1663,1664..1687))
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             complement(1700..1717)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1727..1744)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1763..1785
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1800..1824
                     /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     complement(1824..1843)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1825..1843)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1927..1946)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2152..2229
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2230..3312
                     /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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2249..2268)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4624..4815
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4809..4831)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4917..5056
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4972..4991
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5071..5088)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5242..5830)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5322..5341)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6001..6792,6793..6861))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6624..6643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6862..6966)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7034..7052
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(7079..7084,7085..7094,1..7,8..13)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
ORIGIN
        1 gataagcttt aatgcggtag tttatcacag ttaaattgct aacgcagtca ggcaccgtgt
       61 atgaaatcta acaatgcgct catcgtcatc ctcggcaccg tcaccctgga tgctgtaggc
      121 ataggcttgg ttatgccggt actgccgggc ctcttgcggg atatccggat atagttcctc
      181 ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt
      241 gctcagcggt ggcagcagcc aacttagctt cctttcgggc ctcgagtgcg gccgctcatc
      301 ttttccggct aaatctggcc atgcccggtc tttgtggatt agaaacgaat ccaggatttt
      361 cagcacccgg cttccagctc actttcattt ttgcaccgat actattataa aactgattta
      421 gttcatcaat gttgttggca attgtccatt ttcctttgct aacaatagcg acaggaaatt
      481 caccgggttc agcaacagcc ttgacaatcc ccccgtgttt ttttatttcc tctttttctt
      541 tatccatagc aaaagccggg tagcttacct gggctccatg cctgataaca ttatgctcta
      601 tatctccaaa tggccggttt gaatcagatc gagcaaccaa tcggttaatg cgatctataa
      661 ttttcctttc ctctgatgag ttggatggta ctgaatgagg ttgacctgtg aaactaatca
      721 tatcatgcat atcatagtca ccagtaaaag gtagtgcagc ccagttctct tttgctttca
      781 aggctgagag actcgcttcc agattgttta agtctatagg ataaattttt ccattgacct
      841 gttcatctga taaaccgtga ccactactca tgtaaatacc caaaaggcgg ccagtttttt
      901 tatcccaatg gcctacatag ccttcaatac ccgcttcctg tactttcttt attacatctg
      961 aagcttcgtc tttgggataa gctttgttaa ttgagccggg tttaatggtt ttttccaaaa
     1021 tatcatgccc cttggctgcc gcacctttgc ccaaagctct gagtgttggc ccgcccgcag
     1081 cccgaaaact gacagcaaaa ttacctaatc ttgaagcttc ttcaatctcc ttcatatgtc
     1141 cctcccaaat aaattctctc ttaacttgct gccgaataaa agggtcatca agcgaattat
     1201 ttgagaatga aaggtaccca ctagaaaaat ctgacgatga agatttccca tgtgaatttt
     1261 taatattctg tttggggttt atctgataat tagcatatct aggcatggat ccaccaatct
     1321 gttctctgtg agcctcaata atatcgttat cctccatgtc caaatcttca ggggtctgat
     1381 cagcttggat tctaataccg tcgtacaaga atcttaagga gtccatttcc ttaccctgtc
     1441 ttttagcgaa cgcttccatc agccttctta aaggagtggt ctttttgatc ttgaagaaga
     1501 tctctgaaga tccatcggac acctttaaat tgatgtgagt ctcaggcttg acttctggct
     1561 tgacctctgg cttagcttct tgattgactt ctgagtccga cattttctca aactgagggt
     1621 gtgaccatgc actaccacct gaaccaccac cgctaccacc acctttttca aactgcggat
     1681 ggctccagct agccatatgg ctgccgcgcg gcaccaggcc gctgctgtga tgatgatgat
     1741 gatggctgct gcccatggta tatctccttc ttaaagttaa acaaaattat ttctagaggg
     1801 gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga
     1861 tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
     1921 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
     1981 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
     2041 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac
     2101 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc
     2161 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     2221 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     2281 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     2341 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     2401 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     2461 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     2521 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     2581 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     2641 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     2701 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     2761 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     2821 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     2881 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     2941 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     3001 accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa
     3061 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     3121 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     3181 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     3241 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     3301 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc
     3361 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     3421 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     3481 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg
     3541 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc
     3601 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccgacgcgct
     3661 gggctacgtc ttgctggcgt tcgcgacgcg aggctggatg gccttcccca ttatgattct
     3721 tctcgcttcc ggcggcatcg ggatgcccgc gttgcaggcc atgctgtcca ggcaggtaga
     3781 tgacgaccat cagggacagc ttcaaggatc gctcgcggct cttaccagcc taacttcgat
     3841 cattggaccg ctgatcgtca cggcgattta tgccgcctcg gcgagcacat ggaacgggtt
     3901 ggcatggatt gtaggcgccg ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc
     3961 atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa cggattcacc
     4021 actccaagaa ttggagccaa tcaattcttg cggagaactg tgaatgcgca aaccaaccct
     4081 tggcagaaca tatccatcgc gtccgccatc tccagcagcc gcacgcggcg catctcgggc
     4141 agcgttgggt cctggccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta
     4201 ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga
     4261 agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc
     4321 cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc
     4381 tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc
     4441 tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta
     4501 ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc
     4561 ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca
     4621 tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca
     4681 ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa
     4741 tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac
     4801 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     4861 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca
     4921 gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag
     4981 agcagattgt actgagagtg caccattgcg gtgtgaaata ccgcacagat gcgtaaggag
     5041 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     5101 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     5161 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5221 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5281 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5341 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5401 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5461 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5521 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5581 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5641 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     5701 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5761 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5821 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5881 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5941 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6001 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6061 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6301 cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6601 ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct
     6661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6961 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     7021 attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat tctcatgttt
     7081 gacagcttat catc
//
