LOCUS       sequence-305978-        4016 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes a mini-transposon derived from V. cholerae Tn6677 and T7-GDH
            cargo gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4016)
  AUTHORS   Zhang Y, Sun X, Wang Q, Xu J, Dong F, Yang S, Yang J, Zhang Z, Qian 
            Y, Chen J, Zhang J, Liu Y, Tao R, Jiang Y, Yang J, Yang S
  TITLE     Multicopy chromosomal integration using CRISPR-associated 
            transposases.
  JOURNAL   ACS Synth Biol. 2020 Jun 17. doi: 10.1021/acssynbio.0c00073.
  PUBMED    32551502
REFERENCE   2  (bases 1 to 4016)
  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: "ACS Synth 
            Biol. 2020 Jun 17. doi: 10.1021/acssynbio.0c00073."
COMMENT     Sequence Label: pDonor-GDH
FEATURES             Location/Qualifiers
     source          1..4016
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     116..135
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     369..386
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    503..524
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(539..544,545..562,563..569)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    577..593
                     /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     582..604
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     601..617
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     601..617
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     806..825
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        806..824
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    825..849
                     /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)."
     RBS             864..886
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1689..1706
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1759..1777)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1773..1820
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(2017..2034)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2017..2033)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2026..2048)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2242..2261)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2361..2383
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2421..2439)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2507..2611
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2612..2680,2681..3472)
                     /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(2830..2849)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(3643..4016,1..215)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
       61 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
      121 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
      181 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
      241 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
      301 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
      361 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
      421 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
      481 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
      541 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      601 caggaaacag ctatgaccat gattacgcca agctttgttg atacaaccat aaaatgataa
      661 ttacacccat aaattgataa ttatcacacc cataaattga tattgcctct tcatggtcta
      721 aacttcagta agtttacgac attttcatga agaggtcatt tccggggatc cggcgtagag
      781 gatcgagatc tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat
      841 aacaattccc ctctagaaat aattttgttt aactttaaga aggagatata catatgtatc
      901 cggatttaaa aggaaaagtc gtcgctatta caggagctgc ttcagggctc ggaaaggcga
      961 tggccattcg cttcggcaag gagcaggcaa aagtggttat caactattat agtaataaac
     1021 aagatccgaa cgaggtaaaa gaagaggtca tcaaggcggg cggtgaagct gttgtcgtcc
     1081 aaggagatgt cacgaaagag gaagatgtaa aaaatatcgt gcaaacggca attaaggagt
     1141 tcggcacact cgatattatg attaataatg ccggtcttga aaatcctgtg ccatctcacg
     1201 aaatgccgct caaggattgg gataaagtca tcggcacgaa cttaacgggt gcctttttag
     1261 gaagccgtga agcgattaaa tatttcgtag aaaacgatat caagggaaat gtcattaaca
     1321 tgtccagtgt gcacgaagtg attccttggc cattatttgt ccactatgcg gcaagtaaag
     1381 gcgggatgaa gctgatgaca cgcacattag cgttggaata cgcgccgaag ggcattcgcg
     1441 tcaataatat tgggccaggt gcgatcaaca cgacgatcaa taaggagaaa tttgctgacc
     1501 ctgaacagag agctgatgta gaaagcatga ttccaatggg atatatcggc gaaccggagg
     1561 agatcgccgc agtagcagcc tggcttgctt cgaaggaagc cagctacgtc acaggcatca
     1621 cgttattcgc ggacggcggt atgacacttt atccttcatt ccaggcaggc cgcggttaat
     1681 gactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     1741 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1801 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatctaga gtccttactg
     1861 cagtagtttt gctgaaatac tcgattcaca aaaatatcaa cttatggttg ttttgtgaga
     1921 tatcaatata tggttgtttt gtggttaagt tgctgattat aaataattat taaatatcac
     1981 tttatggttg catcaacagg taccgagctc gaattcactg gccgtcgttt tacaacgtcg
     2041 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     2101 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     2161 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     2221 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     2281 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     2341 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2401 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     2461 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2521 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2581 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     2641 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     2701 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     2761 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     2821 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     2881 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     2941 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3001 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3061 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3121 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     3181 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     3241 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3301 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     3361 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     3421 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     3481 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     3541 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     3601 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     3661 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     3721 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     3781 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     3841 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     3901 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3961 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactga
//
