LOCUS       sequence-433685-        4330 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Use together with with pJE13HD7 and pJE13HD7 derivatives to 
            integrate heterologous genes at ho locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4330)
  AUTHORS   Evans S, Lu Z, McDonnell L, Anderson W, Peralta F, Watkins T , Ahmed
            H, Luna-Flores CH, Loan T, Navone L, Trau M, Scott CH, Speight R, 
            Vickers CE, Peng B
  TITLE     The integration of tandem gene repeats via a bacterial type-II 
            toxin-antitoxin-mediated gene amplification (ToxAmp) system and 
            stability visualisation in Saccharomyces cerevisiae
  JOURNAL   bioRxiv 2024.01.30.578080
REFERENCE   2  (bases 1 to 4330)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.01.30.578080"
COMMENT     Sequence Label: pITEdv1
FEATURES             Location/Qualifiers
     source          1..4330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..29)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13..29)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(26..48)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    37..53
                     /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(61..67,68..85,86..91))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    106..127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(244..261)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(415..1003)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(495..514)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1174..1965,1966..2034))
                     /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     1797..1816
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2035..2139)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2207..2225
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2263..2285)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2385..2404
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2598..2620
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2612..2629
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2613..2629
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2648..3676
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2648..3676
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      3684..3931
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3700..3718)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
       61 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
      121 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
      181 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
      241 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
      301 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
      361 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
      421 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      481 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
      541 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
      601 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
      661 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
      721 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
      781 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
      841 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
      901 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
      961 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     1021 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     1081 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     1141 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     1201 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     1261 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     1321 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     1381 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     1441 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     1501 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     1561 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     1621 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     1681 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     1741 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     1801 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     1861 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     1921 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     1981 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     2041 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     2101 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     2161 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     2221 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     2281 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     2341 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     2401 tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     2461 ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     2521 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     2581 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg catgcgttta
     2641 aacaaccatg ggtaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga
     2701 aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt
     2761 cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt
     2821 ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt
     2881 gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg
     2941 tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga
     3001 ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg
     3061 accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc
     3121 ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc
     3181 tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc
     3241 ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg
     3301 gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc
     3361 gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc
     3421 aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag
     3481 cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt
     3541 ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg
     3601 gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc
     3661 gagggcaaag gaataatctc gagtcatgta attagttatg tcacgcttac attcacgccc
     3721 tccccccaca tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc
     3781 tatttatttt tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct
     3841 tttttttctg tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa
     3901 ggttttggga cgctcgaagg ctttaacgag ctctcgagaa cccttaaagt tacgctaggg
     3961 ataacagggt aatatagtct agacagttaa aggaccagag tgtataaaat gtggcggaat
     4021 cttacagttt gatgctgtcc gcgggcctca taagagttgt ggtaacaacg caggtgcgcg
     4081 catctgctaa aatgtgtata ttagtttaaa aagttgtatg taataaaagt aaaatttaat
     4141 attttggatg aaaaaaagga tccaaacctg caggccattt ttagactttt tcttaactag
     4201 aatgctggag tagaaatacg ccatctcaag atacaaaaag cgttaccggc actgatttgt
     4261 ttcaaccagt atatagatta ttattgggtc ttgatcaact ttcctcagac atatcagtaa
     4321 cagtttaaac
//
