LOCUS       Exported                7996 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pHD-Kan, complete sequence.
ACCESSION   KF947529
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 pHeavens 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)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7996
                     /organism="Cloning vector pHD-Kan"
                     /mol_type="other DNA"
                     /db_xref="taxon:1479043"
     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..1727
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE
                     DPAY"
     CDS             2047..2352
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB"
                     /label=ccdB
                     /protein_id="AHW49186.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     gene            2047..2352
                     /gene="ccdB"
                     /label=ccdB
                     /note="suicide gene"
     intron          2466..2695
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     regulatory      2762..3362
                     /regulatory_class="ribosome_binding_site"
     misc_feature    2778..3351
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             3375..4409
                     /codon_start=1
                     /gene="hyg(R)"
                     /product="hygromycin resistance protein"
                     /label=hyg(R)
                     /note="Hyg(R)"
                     /protein_id="AHW49185.1"
                     /translation="MDRSGKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVG
                     GRGYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQ
                     DLPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSE
                     AMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSL
                     VDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRP
                     RAKE"
     gene            3375..4409
                     /gene="hyg(R)"
                     /label=hyg(R)
     CDS             3390..4409
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /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    5099..5115
                     /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)."
     promoter        complement(5123..5153)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5168..5189
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      5418..6091
     rep_origin      complement(5477..6065)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     polyA_signal    6394..6441
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     CDS             complement(6673..7467)
                     /codon_start=1
                     /gene="kan(R)"
                     /product="kanamycin resistance protein"
                     /label=kan(R)
                     /note="Kan (R)"
                     /protein_id="AHW49187.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     gene            complement(6673..7467)
                     /gene="kan(R)"
                     /label=kan(R)
     promoter        complement(7502..7859)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7516..7651
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(7861..7965)
                     /gene="bla"
                     /label=AmpR promoter