LOCUS       Exported                4631 bp DNA     circular SYN 05-AUG-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4631)
  AUTHORS   Lehti TA, Pajunen MI, Jokilammi A, Korja M, Lilie H, Vettenranta K, 
            Finne J.
  TITLE     Design of a cytotoxic neuroblastoma-targeting agent using an enzyme 
            acting on polysialic acid fused to a toxin.
  JOURNAL   Mol Cancer Ther 2021 Jul 26.
  PUBMED    34315766
REFERENCE   2  (bases 1 to 4631)
  AUTHORS   Finne lab
  TITLE     Direct Submission
  JOURNAL   Exported Aug 5, 2021 from SnapGene 5.3.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1158/1535-7163.MCT-20-1031"; journalName: "Mol Cancer Ther 2021 
            Jul 26."
COMMENT     host strain M15 pREP4
FEATURES             Location/Qualifiers
     source          1..4631
                     /organism="synthetic DNA construct"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
     promoter        6..50
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    26..42
                     /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)."
     protein_bind    58..74
                     /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             97..102
                     /note="ribosome binding site"
     CDS             111..113
                     /codon_start=1
                     /label=start codon
                     /translation="M"
     CDS             123..140
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             150..1307
                     /codon_start=1
                     /gene="Corynebacterium diphtheriae tox"
                     /product="diphtheria toxin fragments A and T
                     >NC_002935.2:188878-190761 Corynebacterium diphtheriae NCTC
                     13129, complete sequence>V01536.1 Corynephage omega gene 
                     tox encoding diphtheria toxin"
                     /label=DT386
                     /translation="GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGN
                     YDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKK
                     ELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVEL
                     EINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKE
                     HGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAW
                     AVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMV
                     AQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKT"
     misc_feature    1312..1358
                     /label=MCS
                     /note="multiple cloning site"
     misc_feature    1358..1368
                     /label=stop codons
                     /note="stop codons in all three reading frames"
     terminator      1374..1468
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1512..2171
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     terminator      2236..2322
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(2803..3391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3562..4422)
                     /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"
     promoter        complement(4423..4527)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agaaatcata aaaaatttat ttgctttgtg agcggataac aattataata gattcaattg
       61 tgagcggata acaatttcac acagaattca ttaaagagga gaaattaact atgagaggat
      121 ctcaccatca ccatcaccat ggcggccgcg gcgctgatga tgttgttgat tcttctaaat
      181 cttttgtgat ggaaaacttt tcttcgtacc acgggactaa acctggttat gtagattcca
      241 ttcaaaaagg tatacaaaag ccaaaatctg gtacacaagg aaattatgac gatgattgga
      301 aagggtttta tagtaccgac aataaatacg acgctgcggg atactctgta gataatgaaa
      361 acccgctctc tggaaaagct ggaggcgtgg tcaaagtgac gtatccagga ctgacgaagg
      421 ttctcgcact aaaagtggat aatgccgaaa ctattaagaa agagttaggt ttaagtctca
      481 ctgaaccgtt gatggagcaa gtcggaacgg aagagtttat caaaaggttc ggtgatggtg
      541 cttcgcgtgt agtgctcagc cttcccttcg ctgaggggag ttctagcgtt gaatatatta
      601 ataactggga acaggcgaaa gcgttaagcg tagaacttga gattaatttt gaaacccgtg
      661 gaaaacgtgg ccaagatgcg atgtatgagt atatggctca agcctgtgca ggaaatcgtg
      721 tcaggcgatc agtaggtagc tcattgtcat gcataaatct tgattgggat gtcataaggg
      781 ataaaactaa gacaaagata gagtctttga aagagcatgg ccctatcaaa aataaaatga
      841 gcgaaagtcc caataaaaca gtatctgagg aaaaagctaa acaataccta gaagaatttc
      901 atcaaacggc attagagcat cctgaattgt cagaacttaa aaccgttact gggaccaatc
      961 ctgtattcgc tggggctaac tatgcggcgt gggcagtaaa cgttgcgcaa gttatcgata
     1021 gcgaaacagc tgataatttg gaaaagacaa ctgctgctct ttcgatactt cctggtatcg
     1081 gtagcgtaat gggcattgca gacggtgccg ttcaccacaa tacagaagag atagtggcac
     1141 aatcaatagc tttatcgtct ttaatggttg ctcaagctat tccattggta ggagagctag
     1201 ttgatattgg tttcgctgca tataattttg tagagagtat tatcaattta tttcaagtag
     1261 ttcataattc gtataatcgt cccgcgtatt ctccggggca taaaacgtaa ggatccgcat
     1321 gcgagctcgg taccccgggt cgacctgcag ccaagcttaa ttagctgagc ttggactcct
     1381 gttgatagat ccagtaatga cctcagaact ccatctggat ttgttcagaa cgctcggttg
     1441 ccgccgggcg ttttttattg gtgagaatcc aagctagctt ggcgagattt tcaggagcta
     1501 aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc
     1561 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg
     1621 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc
     1681 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattt cgtatggcaa
     1741 tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg
     1801 agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc
     1861 tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag
     1921 ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg
     1981 atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt
     2041 atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg
     2101 atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg
     2161 gcggggcgta atttttttaa ggcagttatt ggtgccctta aacgcctggg gtaatgactc
     2221 tctagcttga ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     2281 atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccctc tagagctgcc
     2341 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     2401 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     2461 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     2521 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     2581 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     2641 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     2701 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     2761 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2821 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2881 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2941 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3001 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3061 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3121 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3181 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3241 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3301 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3361 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3421 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3481 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3541 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3601 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     3661 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     3721 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3781 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     3841 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     3901 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     3961 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4021 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4081 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4141 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4201 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4261 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4321 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     4381 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     4441 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4501 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     4561 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     4621 tcttcacctc g
//
