LOCUS       40924_24388        7996 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pHD-Kan, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7996)
  AUTHORS   Schefer Q, Hallmann S, Grotzinger C.
  TITLE     Knockin' on pHeaven's Door: A Fast and Reliable High-Throughput 
            Compatible Zero-Background Cloning Procedure
  JOURNAL   Mol. Biotechnol. 56 (5), 449-458 (2014)
  PUBMED    24499941
REFERENCE   2  (bases 1 to 7996)
  AUTHORS   Grotzinger C, Hallmann S, Schefer Q.
  TITLE     Direct Submission
  JOURNAL   Submitted (09-DEC-2013) Gastroenterology, Charite - University 
            Medicine Berlin, Augustenburger Platz 1, Berlin, Berlin 13353, 
            Germany
REFERENCE   3  (bases 1 to 7996)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7996)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol. 
            Biotechnol."; date: "2014"; volume: "56"; issue: "5"; pages: 
            "449-458"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (09-DEC-2013) Gastroenterology, Charite - University Medicine 
            Berlin, Augustenburger Platz 1, Berlin, Berlin 13353, Germany"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        963..993
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1047..1724
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     CDS             2047..2349
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     intron          2466..2695
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     misc_feature    2778..3364
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             3390..4406
                     /codon_start=1
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     polyA_signal    4725..4949
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5075..5091)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5099..5115)
                     /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)."
     promoter        complement(5123..5153)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5168..5189)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5477..6065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     polyA_signal    complement(6394..6441)
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     CDS             complement(6676..7467)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(7502..7859)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        complement(7861..7965)
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatac ggagagccaa gcttggtacc
      901 gagctcggat ccaagcttgt ttatgtgaga ccgaattcgc ggccgcatta ggcaccccag
      961 gctttacact ttatgcttcc ggctcgtata atgtgtggat tttgagttag gatccgtcga
     1021 gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg
     1081 atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta
     1141 cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata
     1201 agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg
     1261 aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt
     1321 acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg
     1381 atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg
     1441 cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga
     1501 gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca
     1561 ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc
     1621 atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact
     1681 gcgatgagtg gcaggcgggg cgtaatctag aggatccggc ttactaaaag ccagataaca
     1741 gtatgcgtat ttgcgcgctg atttttgcgg tataagaata tatactgata tgtatacccg
     1801 aagtatgtca aaaagaggta tgctatgaag cagcgtatta cagtgacagt tgacagcgac
     1861 agctatcagt tgctcaaggc atatatgatg tcaatatctc cggtctggta agcacaacca
     1921 tgcagaatga agcccgtcgt ctgcgtgccg aacgctggaa agcggaaaat caggaaggga
     1981 tggctgaggt cgcccggttt attgaaatga acggctcttt tgctgacgag aacaggggct
     2041 ggtgaaatgc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg
     2101 gatgtacaga gtgatattat tgacacgccc gggcgacgga tggtgatccc cctggccagt
     2161 gcacgtctgc tgtcagataa agtcccccgt gaactttacc cggtggtgca tatcggggat
     2221 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtgtccgt tatcggggaa
     2281 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc
     2341 tggggaatat aaatgtcagg ctcccttata cacagccagt ctgcaggtcg acggtctcgt
     2401 gaaaacggta ccgggccccc agtgtgctgg aattaattcg ctgtctgcga gggccagctg
     2461 ttggggtgag tactccctct caaaagcggg catgacttct gcgctaagat tgtcagtttc
     2521 caaaaacgag gaggatttga tattcacctg gcccgcggtg atgcctttga gggtggccgc
     2581 gtccatctgg tcagaaaaga caatcttttt gttgtcaagc ttgaggtgtg gcaggcttga
     2641 gatctggcca tacacttgag tgacaatgac atccactttg cctttctctc cacaggtgtc
     2701 cactcccagg tccaactgca ggtcgagcat gcatctaggg cggccgcact agaggaattc
     2761 cgcccctctc ccccccaccc ctctccctcc ccccccccta acgttactgg ccgaagccgc
     2821 ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt
     2881 ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt
     2941 tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
     3001 gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca
     3061 cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg
     3121 gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc
     3181 tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct
     3241 gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
     3301 ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata agcttgccac
     3361 aacccgtacc aaagatggat agatccggaa agcctgaact caccgcgacg tctgtcgaga
     3421 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
     3481 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     3541 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
     3601 cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc
     3661 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     3721 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     3781 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     3841 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     3901 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     3961 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     4021 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     4081 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     4141 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     4201 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     4261 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     4321 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     4381 ccagcactcg tccgagggca aaggaataga gtagatgccg accgaacaag agctgatttc
     4441 gagaacgcct cagccagcaa ctcgcgcgag cctagcaagg caaatgcgag agaacggcct
     4501 tacgcttggt ggcacagttc tcgtccacag ttcgctaagc tcgctcggct gggtcgcggg
     4561 agggccggtc gcagtgattc aggcccttct ggattgtgtt ggtccccagg gcacgattgt
     4621 catgcccacg cactcgggtg atctgactga tcccgcagat tggagatcgc cgcccgtgcc
     4681 tgccgattgg gtgcagatct ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     4741 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     4801 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     4861 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     4921 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     4981 gagtgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5041 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5101 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5161 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5221 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5281 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5341 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5401 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5461 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5521 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5581 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5641 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5701 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5761 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5821 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5881 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5941 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6001 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6061 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6121 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6181 aaaaatgaag ttttaaatca atctaaagta tatatgagta acctgaggct atggcagggc
     6241 ctgccgcccc gacgttggct gcgagccctg ggccttcacc cgaacttggg gggtggggtg
     6301 gggaaaagga agaaacgcgg gcgtattggc cccaatgggc tctcggtggg gtatcgacag
     6361 agtgccagcc ctgggaccga accccgcgtt tatgaacaaa cgacccaaca ccgtgcgttt
     6421 tattctgtct ttttattgcc gtcatagcgc gggttccttc cggtattgtc tccttccgtg
     6481 tttcagttag cctcccccta gggtgggcga agaactccag catgagatcc ccgcgctgga
     6541 ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg
     6601 cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc atttcgaacc
     6661 ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc
     6721 gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc
     6781 agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc
     6841 acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc
     6901 gccatgggtc acgacgagat cctcgccgtc gggcatgctc gccttgagcc tggcgaacag
     6961 ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc
     7021 ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt
     7081 agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc
     7141 aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc
     7201 ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag
     7261 ccacgatagc cgcgctgcct cgtcttgcag ttcattcagg gcaccggaca ggtcggtctt
     7321 gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc
     7381 gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc
     7441 tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct catcctgtct cttgatcgat
     7501 ctttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat agctcagagg
     7561 ccgaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat
     7621 gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg
     7681 ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc
     7741 cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc
     7801 ctggggactt tccacaccct aactgacaca cattccacag ctggttcttt ccgcctcagg
     7861 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     7921 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     7981 agtgccacct gacgtc
//
