LOCUS       62056_8945        5829 bp DNA     circular SYN 28-JUL-2021
DEFINITION  Cloning vector pEGC, complete sequence.
ACCESSION   MZ395121
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5829)
  AUTHORS   Becker K, Meyer A, Roberts TM, Panke S.
  TITLE     Plasmid replication based on the T7 origin of replication requires a
            T7 RNAP variant and inactivation of ribonuclease H
  JOURNAL   Nucleic Acids Res (2021) In press
  PUBMED    34255845
REFERENCE   2  (bases 1 to 5829)
  AUTHORS   Becker K.
  TITLE     Direct Submission
  JOURNAL   Submitted (10-JUN-2021) D-BSSE, ETHZ, Mattenstrasse 26, Basel, BS 
            4058, Switzerland
REFERENCE   3  (bases 1 to 5829)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res (2021) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (10-JUN-2021) D-BSSE, ETHZ, Mattenstrasse 26, Basel, BS 4058, 
            Switzerland"
FEATURES             Location/Qualifiers
     source          1..5829
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      17..605
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(791..933)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     CDS             complement(1038..1226)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     protein_bind    complement(2001..2022)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(2038..3117)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(3118..3195)
                     /label=lacI promoter
     promoter        3504..3522
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3523..3547
                     /label=lac operator
                     /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             3562..3584
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3592..4305
                     /codon_start=1
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Nager et al., 2011)"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             4312..4329
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     CDS             4339..4356
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      4423..4470
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4507..4962
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(5054..5710)
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(5711..5813)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
ORIGIN
        1 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
       61 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
      181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      601 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      661 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      721 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      781 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
      841 tatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
      901 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
      961 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     1021 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     1081 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     1141 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     1201 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     1261 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     1321 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     1381 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     1441 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     1501 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     1561 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     1621 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     1681 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     1741 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     1801 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     1861 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     1921 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     1981 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     2041 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     2101 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     2161 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     2221 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     2281 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     2341 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     2401 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     2461 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     2521 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     2581 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     2641 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     2701 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     2761 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     2821 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     2881 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     2941 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     3001 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     3061 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     3121 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     3181 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     3241 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     3301 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     3361 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     3421 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     3481 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
     3541 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgagcaaa
     3601 ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat
     3661 gggcacaaat tttctgtccg tggagagggt gaaggtgatg ctacaaacgg aaaactcacc
     3721 cttaaattta tttgcactac tggaaaacta cctgttccgt ggccaacact tgtcactact
     3781 ctgacctatg gtgttcaatg cttttcccgt tatccggatc acatgaaacg gcatgacttt
     3841 ttcaagagtg ccatgcccga aggttatgta caggaacgca ctatatcttt caaagatgac
     3901 gggacctaca agacgcgtgc tgaagtcaag tttgaaggtg atacccttgt taatcgtatc
     3961 gagttaaagg gtattgattt taaagaagat ggaaacattc ttggacacaa actcgagtac
     4021 aactttaact cacacaatgt atacatcacg gcagacaaac aaaagaatgg aatcaaagct
     4081 aacttcaaaa ttcgccacaa cgttgaagat ggttccgttc aactagcaga ccattatcaa
     4141 caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta cctgtcgaca
     4201 caatctgtcc tttcgaaaga tcccaacgaa aagcgtgacc acatggtcct tcttgagttt
     4261 gtaactgctg ctgggattac acatggcatg gatgagctct acaaaggatc ccaccaccac
     4321 caccaccact aactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc
     4381 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg
     4441 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg
     4501 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4561 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4621 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4681 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4741 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4801 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4861 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4921 aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcaggtggc acttttcggg
     4981 gaaatgtgcg cggaacccct caggcgtagc aaccaggcgt ttaagggcac caataactgc
     5041 cttaaaaaaa ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca
     5101 ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc agcggcatca
     5161 gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt
     5221 ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga
     5281 aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca
     5341 catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccagagcg
     5401 atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata
     5461 tcaccagctc accgtctttc attgccatac gaaattccgg atgagcattc atcaggcggg
     5521 caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa
     5581 aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg
     5641 cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt
     5701 ttttctccat tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg
     5761 gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc
     5821 attttcgcc
//
