LOCUS       sequence-375095-        3009 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A basic cloning vector for inserting genes with 5' and 3' UTRs for 
            later subcloning via Golden Gate assembly..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3009)
  AUTHORS   Hall RN, Weill U, Drees L, Leal-Ortiz S, Li H, Khariton M, Chai C, 
            Xue Y, Rosental B, Quake SR, Sanchez Alvarado A, Melosh NA, Fire AZ,
            Rink JC, Wang B
  TITLE     Heterologous reporter expression in the planarian Schmidtea 
            mediterranea through somatic mRNA transfection.
  JOURNAL   Cell Rep Methods. 2022 Sep 20;2(10):100298. doi: 
            10.1016/j.crmeth.2022.100298. eCollection 2022 Oct 24.
  PUBMED    36313809
REFERENCE   2  (bases 1 to 3009)
  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: "Cell Rep 
            Methods."; date: "2022-09-20"; volume: "2(10):100298. doi"; pages: "
            10.1016/j.crmeth.2022.100298. eCollection 2022 Oct 24"
COMMENT     Sequence Label: pNHT7
FEATURES             Location/Qualifiers
     source          1..3009
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        554..572
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    701..722
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(737..742,743..760,761..767)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    775..791
                     /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     780..802
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     799..815
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             811..1134
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    828..884
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(885..901)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1110..1129)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1229..1251
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1274..1290)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1392..1411)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1403..2212
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1470..1489)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2359..2947
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2848..2867
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agttaatacg actcactata ggagagaccg cgaggaagcg gaagagcgcc
      601 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
      661 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
      721 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
      781 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cttgcatgcc
      841 tgcaggtcga ctctagagga tccccgggta ccgagctcga attcactggc cgtcgtttta
      901 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
      961 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     1021 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     1081 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     1141 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     1201 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     1261 ggtctctaag cttgtcatag ctgtttcctg gcagctctgg cccgtgtctc aaaatctctg
     1321 atgttacatt gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat
     1381 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt
     1441 aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca
     1501 atcaggtgcg acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa
     1561 acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct
     1621 gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg
     1681 gttactcacc actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga
     1741 ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc
     1801 tgtttgtaat tgtcctttta acagcgatcg cgtatttcgc ctcgctcagg cgcaatcacg
     1861 aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt
     1921 tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac
     1981 tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat
     2041 tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg
     2101 cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa
     2161 tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt
     2221 ggttaattgg ttgtaacact ggcagagcat tacgctgact tgacgggacg gcgcaagctc
     2281 atgaccaaaa tcccttaacg tgagttacgc gtcgttccac tgagcgtcag accccgtaga
     2341 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2401 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2461 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     2521 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2581 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2641 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     2701 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     2761 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     2821 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     2881 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     2941 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3001 tcacatgtt
//
